Method for identifying alzheimer&#39;s disease therapeutics using transgenic animal models

ABSTRACT

The construction of transgenic animal models of human Alzheimer&#39;s disease, and methods of using the models to screen potential Alzheimer&#39;s disease therapeutics, are described. The models are characterized by pathologies similar to pathologies observed in Alzheimer&#39;s disease, based on expression of all three forms of the β-amyloid precursor protein (APP), APP695, APP751, and APP770, as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer&#39;s disease (FAD) mutations at amino acid 717, predicted mutations in the APP gene, and truncated forms of APP that contain the Aβ region. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or electroporation. The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer&#39;s disease as measured by their effect on the amount of APP, β-amyloid peptide, and numerous other Alzheimer&#39;s disease markers in the animals, the neuropathology of the animals, as well as by behavioral alterations in the animals.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/480,653.

BACKGROUND OF THE INVENTION

[0002] Transgenic animal models of Alzheimer's disease are describedalong with a method of using the transgenic animal models to screen fortherapeutics useful for the treatment of Alzheimer's disease.

[0003] Alzheimer's disease (AD) is a degenerative disorder of the brainfirst described by Alios Alzheimer in 1907 after examining one of hispatients who suffered drastic reduction in cognitive abilities and hadgeneralized dementia (The early story of Alzheimer's Disease, edited byBick et al. (Raven Press, New York 1987)). It is the leading cause ofdementia in elderly persons. AD patients have increased problems withmemory loss and intellectual functions which progress to the point wherethey cannot function as normal individuals. With the loss ofintellectual skills the patients exhibit personality changes, sociallyinappropriate actions and schizophrenia (A Guide to the Understanding ofAlzheimer's Disease and Related Disorders, edited by Jorm (New YorkUniversity Press, New York 1987). AD is devastating for both victims andtheir families, for there is no effective palliative or preventivetreatment for the inevitable neurodegeneration.

[0004] The impact of AD on society and on the national economy isenormous. It is expected that the demented elderly population in theUnited States will increase by 41% by the year 2000. It is expensive forthe health care systems that must provide institutional and ancillarycare for the AD patients at an estimated annual cost of $40 billion(Jorm (1987); Fisher, “Alzheimer's Disease”, New York Times, Aug. 23,1989, page D1, edited by Reisberg (The Free Press, New York & London1983)). These factors imply action must be taken to generate effectivetreatments for AD.

[0005] At a macroscopic level, the brains of AD patients are usuallysmaller, sometimes weighing less than 1,000 grams. At a microscopiclevel, the histopathological hallmarks of AD include neurofibrillarytangles (NFT), neuritic plaques, and degeneration of neurons. ADpatients exhibit degeneration of nerve cells in the frontal and temporalcortex of the cerebral cortex, pyramidal neurons of hippocampus, neuronsin the medial, medial central, and cortical nuclei of the amygdala,noradrenergic neurons in the locus coeruleus, and the neurons in thebasal forebrain cholinergic system. Loss of neurons in the cholinergicsystem leads to a consistent deficit in cholinergic presynaptic markersin AD (Fisher (1983); Alzheimer's Disease and Related Disorders,Research and Development edited by Kelly (Charles C. Thomas,Springfield, Ill. 1984)). In fact, AD is defined by the neuropathologyof the brain.

[0006] AD is associated with neuritic plaques measuring up to 200 μm indiameter in the cortex, hippocampus, subiculum, hippocampal gyrus, andamygdala. One of the principal constituents of neuritic plaques isamyloid, which is stained by Congo Red (Fisher (1983); Kelly (1984)).Amyloid plaques stained by Congo Red are extracellular, pink orrust-colored in bright field, and birefringent in polarized light. Theplaques are composed of polypeptide fibrils and are often present aroundblood vessels, reducing blood supply to various neurons in the brain.

[0007] Various factors such as genetic predisposition, infectiousagents, toxins, metals, and head trauma have all been suggested aspossible mechanisms of AD neuropathy. However, available evidencestrongly indicates that there are distinct types of geneticpredispositions for AD. First, molecular analysis has provided evidencefor mutations in the amyloid precursor protein (APP) gene in certainAD-stricken families (Goate et al. Nature 349:704-706 (1991); Murrell etal. Science 254:97-99 (1991); Chartier-Harlin et al. Nature 353:844-846(1991); Mullan et al., Nature Genet. 1:345-347 (1992)). Additional genesfor dominant forms of early onset AD reside on chromosome 14 andchromosome 1 (Rogaev et al., Nature 376:775-778 (1995); Levy-Lahad etal., Science 269:973-977 (1995); Sherrington et al., Nature 375:754-760(1995)). Another loci associated with AD resides on chromosome 19 andencodes a variant form of apolipoprotein E (Corder, Science 261:921-923(1993).

[0008] Amyloid plaques are abundantly present in AD patients and inDown's Syndrome individuals surviving to the age of 40. Theoverexpression of APP in Down's Syndrome is recognized as a possiblecause of the development of AD in Down's patients over thirty years ofage (Rumble et al., New England J. Med. 320:1446-1452 (1989); Mann etal., Neurobiol. Aging 10:397-399 (1989)). The plaques are also presentin the normal aging brain, although at a lower number. These plaques aremade up primarily of the amyloid β peptide (Aβ; sometimes also referredto in the literature as β-amyloid peptide or β peptide) (Glenner andWong, Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is alsothe primary protein constituent in cerebrovascular amyloid deposits. Theamyloid is a filamentous material that is arranged in beta-pleatedsheets. Aβ is a hydrophobic peptide comprising up to 43 amino acids. Thedetermination of its amino acid sequence led to the cloning of the APPcDNA (Kang et al., Nature 325:733-735 (1987); Goldgaber et al., Science235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190-4194(1987); Tanzi et al., Nature 331:528-530 (1988)) and genomic APP DNA(Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai et al.,Gene 87, 257-263 (1990)). A number of forms of APP cDNA have beenidentified, including the three most abundant forms, APP695, APP751, andAPP770. These forms arise from a single precursor RNA by alternatesplicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al.(1990)). APP contains an extracellular domain, a transmembrane regionand a cytoplasmic domain. Aβ consists of up to 28 amino acids justoutside the hydrophobic transmembrane domain and up to 15 residues ofthis transmembrane domain. Thus, Aβ is a cleavage product derived fromAPP which is normally found in brain and other tissues such as heart,kidney and spleen. However, Aβ deposits are usually found in abundanceonly in the brain.

[0009] The larger alternate forms of APP (APP751, APP770) consist ofAPP695 plus one or two additional domains. APP751 consists of all 695amino acids of APP695 plus an additional 56 amino acids which hashomology to the Kunitz family of serine protease inhibitors (KPI) (Tanziet al. (1988); Weidemann et al., Cell 57:115-126 (1989); Kitaguchi etal., Nature 331:530-532 (1988); Tanzi et al., Nature 329:156 (1987)).APP770 contains all 751 amino acids of APP751 and an additional 19 aminoacid domain homologous to the neuron cell surface antigen OX-2(Weidemann et al. (1989); Kitaguchi et al. (1988)). Unless otherwisenoted, the amino acid positions referred to herein are the positions asthey appear in APP770. The amino acid number of equivalent positions inAPP695 and APP751 differ in some cases due to the absence of the OX-2and KPI domains. By convention, the amino acid positions of all forms ofAPP are referenced by the equivalent positions in the APP770 form.Unless otherwise noted, this convention is followed herein. Unlessotherwise noted, all forms of APP and fragments of APP, including allforms of Aβ, referred to herein are based on the human APP amino acidsequence. APP is post-translationally modified by the removal of theleader sequence and by the addition of sulfate and sugar groups.

[0010] Van Broeckhaven et al., Science 248:1120-1122 (1990), havedemonstrated that the APP gene is tightly linked to hereditary cerebralhemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This wasconfirmed by the finding of a point mutation in the APP coding region intwo Dutch patients (Levy et al., Science 248:1124-1128 (1990)). Themutation substituted a glutamine for glutamic acid at position 22 of theAβ (position 618 of APP695, or position 693 of APP770). In addition,certain families are genetically predisposed to Alzheimer's disease, acondition referred to as familial Alzheimer's disease (FAD), throughmutations resulting in an amino acid replacement at position 717 of thefull length protein (Goate et al. (1991); Murrell et al. (1991);Chartier-Harlin et al. (1991)). These mutations co-segregate with thedisease within the families and are absent in families with late-onsetAD. This mutation at amino acid 717 increases the production of theAβ₁₋₄₂ form of Aβ from APP (Suzuki et al., Science 264.1336-1340(1994)). Another mutant form contains a change in amino acids atpositions 670 and 671 of the full length protein (Mullan et al. (1992)).This mutation to amino acids 670 and 671 increases the production oftotal Aβ from APP (Citron et al., Nature 360:622-674 (1992)).

[0011] There are no robust animal models to study AD, although agingnonhuman primates seem to develop amyloid plaques of Aβ in brainparenchyma and in the walls of some meningeal and cortical vessels.Although aged primates and canines can serve as animal models, they areexpensive to maintain, need lengthy study periods, and are quitevariable in the extent of pathology that develops.

[0012] There are no spontaneous animal mutations with sufficientsimilarities to AD to be useful as experimental models. Various modelshave been proposed in which some AD-like symptoms may be induced byelectrolysis, transplantation of AD brain samples, aluminum chloride,kainic acid or choline analogs (Kisner et al., Neurobiol. Aging7:287-292 (1986); Mistry et al., J Med Chem 29:337-343 (1986)). Flood etal., Proc. Natl. Acad. Sci. 88:3363-3366 (1986), reported amnesticeffects in mice of four synthetic peptides homologous to the Aβ. Becausenone of these share with AD either common symptoms, biochemistry orpathogenesis, they are not likely to yield much useful information onetiology or treatment.

[0013] Several transgenic rodent lines have been produced that expresseither the human APP gene or human APP complementary DNA regulated by avariety of promoters. Transgenic mice with the human APP promoter linkedto E. coli β-galactosidase (Wirak et al., The EMBO J 10:289-296 (1991))as well as transgenic mice expressing the human APP751 cDNA (Quon et al.Nature 352:239-241 (1991)) or subfragments of the cDNA including the Aβ(Wirak et al., Science 253:323-325 (1991); Sandhu et al., J. Biol. Chem.266:21331-21334 (1991); Kawabata et al., Nature 354:476-478 (1991)) havebeen produced. Results obtained in the different studies appear todepend upon the source of promoter and the protein coding sequence used.For example, Wirak et al., Science 253:323-325 (1991), found that intransgenic mice expressing a form of the Aβ, intracellular accumulationof “amyloid-like” material, reactive with antibodies prepared against Aβwere observed but did not find other histopathological disease symptoms.The intracellular nature of the antibody-reactive material and the lackof other symptoms suggest that this particular transgenic animal is nota faithful model system for Alzheimer's disease. Later studies haveshown that similar staining is seen in non-transgenic control mice andWirak et al., Science 253:323-325 (1991) was partially retracted in acomment in Science 255:143-145 (1992). Thus, the staining seen by Wiraket al. appears to be artifactual.

[0014] Kawabata et al. (1991) report the production of amyloid plaques,neurofibrillary tangles, and neuronal cell death in their transgenicanimals. In each of these studies, Aβ or a fragment containing Aβ wasexpressed. Wirak et al. (1991), used the human APP promoter whileKawabata et al. (1991) used the human thy-1 promoter. However, Kawabataet al. (1991) was later retracted by Kawabata et al., Nature 356:23(1992) and Kawabata et al., Nature 356:265 (1992). In transgenic miceexpressing the APP751 cDNA from the neuron-specific enolase promoter ofQuon et al. (1991), rare, small extracellular deposits of materialreactive with antibody prepared against synthetic Aβ were observed. Areview of the papers describing these early transgenic mice indicatethat do not produce characteristic Alzheimer pathologies (see Marx,Science 255:1200-1202 (1992)).

[0015] Transgenic mice expressing APP751 from a neuron-specific enolase(NSE) promoter were recently described by McConlogue et al., Neurobiol.Aging 15:S12 (1994), Higgins et al., Ann Neurol. 35:598-607 (1995),Mucke et al., Brain Res. 666:151-167 (1994), Higgins et al., Proc. Natl.Acad. Sci. USA 92:4402-4406 (1995), and U.S. Pat. No. 5,387,742 toCordell. Higgins et al., Ann Neurol. 35:598-607 (1995) describe resultswith the same mice as described by Quon et al. (1991). Such mice haveonly sparse Aβ deposits which are more typical of very early AD andyoung Down's syndrome cases. The deposits seen in this transgenic mousewere also seen, although at a lower abundance, in non-transgenic controlanimals. Mature lesions such as frequent compacted plaques, neuriticdystrophy and extensive gliosis are not seen in these mice (Higgins etal., Ann Neurol. 35:598-607 (1995)). McConlogue et al. (1994) reportedfinding no Aβ deposits in these mice.

[0016] Transgenic mice in which APP is expressed from the neuronalspecific synaptophysin promoter express APP at low levels equivalent tothat in brain tissue from the NSE APP mice described above. These micewere also reported not to display any brain lesions (Higgins et al.).

[0017] Transgenic mice containing yeast artificial chromosome (YAC) APPconstructs have also been made (Pearson and Choi, Proc. Natl. Acad. Sci.USA 90:10578-10582 (1993); Lamb et al., Nature Genetics 5:22-30 (1993);Buxbaum et al., Biochem. Biophys. Res. Comm. 197:639-645 (1993)). Thesemice contain the entire human APP genomic gene and express human APPprotein at levels similar to endogenous APP; higher levels of expressionthan that obtained in mice using the NSE promoter. None of these mice,however, show evidence of pathology similar to AD.

[0018] Alzheimer's disease animal models, including transgenic models,have been recently reviewed by Lannfelt et al., Behavioural Brain Res.57:207-213 (1993), and Fukuchi et al., Ann. N. Y. Acad. Sci. 695:217-223(1993). Lannfelt et al. points out that none of the prior transgenicanimals that show apparent plaques demonstrate neuropathological changescharacteristic of AD. Lannfelt et al. also discusses possible reasonsfor the “failure” of previous transgenic animal models. Similarly,Fukuchi et al. discusses the failure of prior transgenic animal modelsto display most of the characteristics known to be associated with AD.For example, the transgenic mouse reported by Quon et al. is reported toproduce Aβ immunoreactive deposits that stain only infrequently withthioflavin S and not at all with Congo Red, in contrast to the stainingpattern of AD Aβ deposits.

[0019] Alzheimer's disease is characterized by numerous changes in theexpression levels of various proteins, the biochemical activity andhistopathology of brain tissue, as well as cognitive changes in affectedindividuals. Such characteristic changes associated with AD have beenwell documented. The most prominent change, as noted above, is thedeposition of Aβ into amyloid plaques (Haass and Selkoe, Cell75:1039-1042 (1993)). A variety of other molecules are also present inplaques, such as apolipoprotein E, laminin, amyloid P component, andcollagen type IV (Kalaria and Perry, Brain Research 631:151-155 (1993);Ueda et al., Proc. Natl. Aca. Sci. USA 90:11282-11286 (1993)). Changesin cytoskeletal markers have also been associated with AD, such as thechanges in microtubule-associated protein tau, MAP-2 or neurofilaments(Kosik et al., Science 256: 780-783 (1992); Lovestone and Anderton,Current Opinion in Neurology & Neurosurgery 5:883-888 (1992); Brandanand Inestrosa, General Pharmacology 24:1063-1068 (1993); Trojanowski etal., Brain Pathology 3:45-54 (1993); Masliah et al., American Journal ofPathology 142:871-882 (1993)). Alzheimer's disease is also known tostimulate an immunoinflammatory response, increasing such inflammatorymarkers as glial fibrillary acidic protein (GFAP), α2-macroglobulin, andinterleukins 1 and 6 (IL-1 and IL-6) (Frederickson and Brunden,Alzheimer Disease and Associated Disorders 8:159-165 (1994); McGeer etal., Canadian Journal of Neurological Sciences 18:376-379 (1991); Woodet al., Brain Research 629:245-252 (1993)). Finally, neuronal andneurotransmitter changes have been associated with AD, such as thecholinergic, muscarinic, serotinergic, adrenergic, and adensosinereceptor systems (Rylett et al., Brain Res 289:169-175 (1983); Sims etal., Lancet 1:333-336 (1980); Nitsch et al., Science 258:304-307 (1992);Masliah and Terry, Clinical Neuroscience 1:192-198 (1993); Greenamyreand Maragos, Cerebrovascular and Brain Metabolism Reviews 5:61-94(1993); McDonald and Nemeroff, Psychiatric Clinics of North America14:421-422 (1991); Mohr et al., Journal of Psychiatry & Neuroscience19:17-23 (1994)).

[0020] It is therefore an object of the present invention to provide ananimal model for Alzheimer's disease that is constructed usingtransgenic technology.

[0021] It is a further object of the present invention to providetransgenic animals characterized by certain genetic abnormalities in theexpression of the amyloid precursor protein.

[0022] It is a further object of the present invention to providetransgenic animals exhibiting one or more histopathologies similar tothose of Alzheimer's disease.

[0023] It is a further object of the present invention to providetransgenic animals expressing one or more Aβ-containing proteins at highlevels in brain tissue.

[0024] It is a further object of the present invention to provide amethod of screening potential drugs for the treatment of Alzheimer'sdisease using transgenic animal models.

SUMMARY OF THE INVENTION

[0025] The construction of transgenic animal models for testingpotential treatments for Alzheimer's disease is described. The modelsare characterized by a greater similarity to the conditions existing innaturally occurring Alzheimer's disease, based on the ability to controlexpression of one or more of the three major forms of the β-amyloidprecursor protein (APP), APP695, APP751, and APP770, or subfragmentsthereof, as well as various point mutations based on naturally occurringmutations, such as the FAD mutations at amino acid 717, and predictedmutations in the APP gene. The APP gene constructs are prepared usingthe naturally occurring APP promoter of human, mouse, or rat origin,efficient promoters such as human platelet derived growth factor β chain(PDGF-B) gene promoter, as well as inducible promoters such as the mousemetallothionine promoter, which can be regulated by addition of heavymetals such as zinc to the animal's water or diet. Neuron-specificexpression of constructs can be achieved by using the rat neuronspecific enolase promoter.

[0026] The constructs are introduced into animal embryos using standardtechniques such as microinjection or embryonic stem cells. Cell culturebased models can also be prepared by two methods. Cells can be isolatedfrom the transgenic animals or prepared from established cell culturesusing the same constructs with standard cell transfection techniques.

[0027] The constructs disclosed herein generally encode all or acontiguous portion of one of the three forms of APP: APP695, APP751, orAPP770, preferably an Aβ-containing protein, as described herein.Examples of Aβ-containing proteins are proteins that include all or acontiguous portion of APP770, APP770 bearing a mutation in amino acid669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutationin amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717,where each of these Aβ-containing proteins includes amino acids 672 to714 of human APP. Some specific constructs that are described employ thefollowing protein coding sequences: the APP770 cDNA; the APP770 cDNAbearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or acombination of these mutations; the APP751 cDNA containing the KPIprotease inhibitor domain without the OX-2 domain in the construct; theAPP751 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692,717, or a combination of these mutations; the APP695 cDNA; the APP695cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or acombination of these mutations; APP695, APP751, or APP770 cDNA truncatedat amino acid 671 or 685, the sites of β-secretase or α-secretasecleavage, respectfully; APP cDNA truncated to encode amino acids 646 to770 of APP; APP cDNA truncated to encode amino acids 646 to 770 of APPand including at least one intron; the APP leader sequence followed bythe Aβ region (amino acids 672 to 714 of APP) plus the remaining carboxyterminal 56 amino acids of APP; the APP leader sequence followed by theAβ region plus the remaining carboxy terminal 56 amino acids with theaddition of a mutation at amino acid 717; the APP leader sequencefollowed by the Aβ region; the Aβ region plus the remaining carboxyterminal 56 amino acids of APP; the Aβ region plus the remaining carboxyterminal 56 amino acids of APP with the addition of a mutation at aminoacid 717; a combination cDNA/genomic APP gene construct; a combinationcDNA/genomic APP gene construct with the addition of a mutation at aminoacid 669, 670, 671, 690, 692, 717, or a combination of these mutations;a combination cDNA/genomic APP gene construct truncated at amino acid671 or 685; and an APP cDNA construct containing at least amino acids672 to 722 of APP.

[0028] These protein coding sequences are operably linked to leadersequences specifying the transport and secretion of the encoded Aβrelated protein. A preferred leader sequence is the APP leader sequence.These combined protein coding sequences are in turn operably linked to apromoter that causes high expression of Aβ in transgenic animal braintissue. A preferred promoter is the human platelet derived growth factorβ chain (PDGF-B) gene promoter. Additional constructs include a humanyeast artificial chromosome construct controlled by the PDGF-B promoter;a human yeast artificial chromosome construct controlled by the PDGF-Bpromoter with the addition of a mutation at amino acid 669, 670, 671,690, 692, 717, or a combination of these mutations; the endogenous mouseor rat APP gene modified through the process of homologous recombinationbetween the APP gene in a mouse or rat embryonic stem (ES) cell and avector carrying the human APP cDNA bearing a mutation at amino acidposition 669, 670, 671, 690, 692, 717, or a combination of thesemutations, such that sequences in the resident rodent chromosomal APPgene beyond the recombination point (the preferred site forrecombination is within APP exon 9) are replaced by the analogous humansequences bearing a mutation at amino acid 669, 670, 671, 690, 692, 717,or a combination of these mutations. These constructs can be introducedinto the transgenic animals and then combined by mating of animalsexpressing the different constructs.

[0029] The transgenic animals, or animal cells, are used to screen forcompounds altering the pathological course of Alzheimer's disease asmeasured by their effect on the amount and/or histopathology ofAlzheimer's disease markers in the animals, as well as by behavioralalterations. These markers include APP and APP cleavage products; Aβ;other plaque related molecules such as apolipoprotein E, laminin, andcollagen type IV; cytoskeletal markers, such as spectrin, tau,neurofilaments, and MAP-2; inflammatory markers, such as GFAP,α2-macroglobulin, IL-1, and IL-6; and neuronal and synapticneurotransmitter related markers, such as GAP43 and synaptophysin, andthose associated with the cholinergic, muscarinic, serotinergic,adrenergic, and adensosine receptor systems.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The boxed portions of the drawings indicate the amino acid codingportions of the constructs. Filled portions indicate the various domainsof the protein as indicated in the Figure Legend. Lines indicatesequences in the clones that are 5′ or 3′ untranslated sequences,flanking genomic sequences, or introns. The break in the line to theleft of the constructs in FIGS. 7 and 8 indicates the presence of a longDNA sequence.

[0031]FIG. 1a is a schematic of the APP770 cDNA coding sequence.

[0032]FIG. 1b is a schematic of the APP770 cDNA coding sequence bearinga mutation at position 717.

[0033]FIG. 2a is a schematic of the APP751 cDNA coding sequence.

[0034]FIG. 2b is a schematic of the APP751 cDNA coding sequence bearinga mutation at position 717.

[0035]FIG. 3a is a schematic of the APP695 coding sequence.

[0036]FIG. 3b is a schematic of the APP695 cDNA coding sequence bearinga mutation at position 717.

[0037]FIG. 4a is a schematic of a coding sequence for the carboxyterminal portion of APP.

[0038]FIG. 4b is a schematic of a coding sequence for the carboxyterminal portion of APP bearing a mutation at position 717.

[0039]FIG. 5 is a schematic of a coding sequence for the Aβ portion ofAPP.

[0040]FIG. 6a is a schematic of a combination cDNA/genomic codingsequence allowing alternative splicing of the KPI and OX-2 exons.

[0041]FIG. 6b is a schematic of a combination cDNA/genomic codingsequence bearing a mutation at position 717 and allowing alternativesplicing of the KPI and OX-2 exons.

[0042]FIG. 7a is a schematic of a human APP YAC coding sequence.

[0043]FIG. 7b is a schematic of a human APP YAC coding sequence bearinga mutation at position 717.

[0044]FIGS. 8a and 8 b are schematics of genetic alteration of the mouseAPP gene by homologous recombination between the mouse APP gene in amouse ES cell and a vector carrying the human APP cDNA (either of thewild-type (FIG. 8a) or FAD mutant form (FIG. 8b)) directed to the exon 9portion of the gene. As a result of this recombination event, sequencesin the resident mouse chromosomal APP gene beyond the recombinationpoint in exon 9 are replaced by the analogous human sequences.

[0045]FIG. 9 is a schematic map of the PDAPP vector, a combinationcDNA/genomic APP construct.

[0046]FIG. 10 is a diagram of the genomic region of APP present in thePDAPP construct. The sizes of original introns 6, 7 and 8, as well asthe sizes of the final introns are indicated on the diagram. Thelocations of the deletions in introns 6 and 8 present in the PDAPPconstruct are also indicated.

[0047]FIG. 11 is a diagram of the intermediate constructs used toconstruct the APP splicing cassette and the PDAPP vector.

[0048]FIG. 12 is a diagram of the PDAPP-wt vector and the plasmids usedto make the PDAPP-wt vector.

[0049]FIG. 13 is a diagram of the PDAPP-Sw/Ha vector and the plasmidsand intermediate constructs used to make the PDAPP-Sw/Ha vector.

[0050]FIG. 14 is a diagram of the PDAPP695_(V-F) vector and the plasmidsand intermediate constructs used to make the PDAPP695_(V-F) vector.

[0051]FIG. 15 is a diagram of the PDAPP751_(V-F) vector and the plasmidsand intermediate constructs used to make the PDAPP751_(V-F) vector.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The constructs and transgenic animals and animal cells areprepared using the methods and materials described below.

[0053] Sources of Materials.

[0054] Restriction endonucleases are obtained from conventionalcommercial sources such as New England Biolabs (Beverly, Mass.), PromegaBiological Research Products (Madison, Wis.), and Stratagene (La JollaCalif.). Radioactive materials are obtained from conventional commercialsources such as Dupont/NEN or Amersham. Custom-designed oligonucleotidesfor site-directed mutagenesis are available from any of severalcommercial providers of such materials such as Bio-Synthesis Inc.,Lewisville, Tex. Kits for carrying out site-directed mutagenesis areavailable from commercial suppliers such as Promega Biological ResearchProducts and Stratagene. Clones of cDNA including the APP695, APP751,and APP770 forms of APP mRNA were obtained directly from Dr. DmitryGoldgaber, NIH. Libraries of DNA are available from commercial providerssuch as Stratagene, La Jolla, Calif., or Clontech, Palo Alto, Calif.PC12 and 3T3 cells were obtained from ATCC (#CRL1721 and #CCL92,respectively). An additional PC12 cell line was obtained from Dr.Charles Marotta of Harvard Medical School, Massachusetts GeneralHospital, and McLean Hospital. Standard cell culture media appropriateto the cell line are obtained from conventional commercial sources suchas Gibco/BRL. Murine stem cells, strain D3, were obtained from Dr. RolfKemler (Doetschman et al., J. Embryol. Exp. Morphol. 87:27 (1985)).Lipofectin for DNA transfection and the drug G418 for selection ofstable transformants are available from Gibco/BRL.

[0055] Definition of APP cDNA Clones.

[0056] The cDNA clone APP695 is of the form of cDNA described by Kang etal., Nature 325:733-735 (1987), and represents the most predominant formof APP in the brain. The cDNA clone APP751 is of the form described byPonte et al., Nature 331:525-527 (1988). This form contains an insert of168 nucleotides relative to the APP695 cDNA. The 168 nucleotide insertencodes the KPI domain. The cDNA clone APP770 is of the form describedby Kitaguchi et al. Nature 331:530-532 (1988). This form contains aninsert of 225 nucleotides relative to the APP695 cDNA. This insertincludes the 168 nucleotides present in the insert of the APP751 cDNA,as well as an addition 57 nucleotide region that does not appear inAPP751 cDNA. The 225 nucleotide insert encodes for the KPI domain aswell as the OX-2 domain. All three forms arise from the same precursorRNA transcript by alternative splicing. The 168 nucleotide insert ispresent in both APP751 cDNA and APP770 cDNA.

[0057] The sequence encoding APP695 is shown in SEQ ID NO:1. Thissequence begins with the first base of the initiation codon AUG andencodes a 695 amino acid protein. The region from nucleotide 1789 to1917 of SEQ ID NO:1 encodes the Aβ. The amino acid sequence of APP695 isshown in SEQ ID NO:2. Amino acids 597 to 639 of SEQ ID NO:2 form the Aβ.The amino-acid composition of the APP695 is A57, C12, D47, E85, F17,G31, H25, I23, K38, L52, M21, N28, P31, Q33, R33, S30, T45, V62, W8, Y17resulting in a calculated molecular weight of 78,644.45. These sequencesare derived from Kang et al. (1988).

[0058] The sequence encoding APP751 is shown in SEQ ID NO:3. Thissequence begins with the first base of the initiation codon AUG andencodes a 751 amino acid protein. Nucleotides 866 to 1033 of SEQ ID NO:3do not appear in APP695 cDNA. The region from nucleotide 1957 to 2085 ofSEQ ID NO:3 encodes the Aβ. The amino acid sequence of APP751 is shownin SEQ ID NO:4. Amino acids 289 to 345 of SEQ ID NO:4 do not appear inAPP695. This 57 amino acid region includes the KPI domain. Amino acids653 to 695 of SEQ ID NO:4 form the Aβ. These sequences are derived fromPonte et al. (1988).

[0059] The sequence encoding APP770 is shown in SEQ ID NO:5. Thissequence begins with the first base of the initiation codon AUG andencodes a 770 amino acid protein. Nucleotides 866 to 1090 of SEQ ID NO:5do not appear in APP695 cDNA. Nucleotides 1034 to 1090 of SEQ ID NO:5 donot appear in APP751 cDNA. The region from nucleotide 2014 to 2142encodes the Aβ. The amino acid sequence of APP770 is shown in SEQ IDNO:6. Amino acids 289 to 364 of SEQ ID NO:6 do not appear in APP695.This 76 amino acid region includes the KPI and OX-2 domains. Amino acids345 to 364 of SEQ ID NO:6 do not appear in APP751. This 20 amino acidregion includes the OX-2 domain. Amino acids 672 to 714 form the Aβ. Aprobable membrane-spanning region of the APP occurs from amino acid 700to 723. Unless otherwise stated, all references herein to nucleotidepositions refer to the numbering of SEQ ID NO:5. This is the numberingderived from the APP770 cDNA. Unless otherwise stated, all referencesherein to amino acid positions refer to the numbering of SEQ ID NO:6.This is the numbering derived from APP770. According to this numberingconvention, for example, amino acid position 717 refers to amino acid717 of APP770, amino acid 698 of APP751, and amino acid 642 of APP695.The above sequences are derived from Kang et al. (1988) and Kitaguchi etal. (1988).

[0060] Unless otherwise noted, all forms of APP and fragments of APP,including all forms of Aβ, referred to herein are based on the human APPamino acid sequence. For example, Aβ refers to the human Aβ, APP refersto human APP, and APP770 refers to human APP770. As used herein, theterm cDNA refers not only to DNA molecules actually prepared by reversetranscription of mRNA, but also any DNA molecule encoding a proteinwhere the coding region is not interrupted, that is, a DNA moleculehaving a continuous open reading frame encoding a protein. As such, theterm cDNA as used herein provides a convenient means of referring to aprotein encoding DNA molecule where the protein encoding region is notinterrupted by intron sequences (or any other sequences not encodingprotein).

[0061] Definition of the APP Genomic Locus.

[0062] Characterization of phage and cosmid clones of human genomic DNAclones listed in Table 1 below originally established a minimum size ofat least 100 kb for the Alzheimer's gene. There are a total of 18 exonsin the APP gene (Lemaire et al., Nucl. Acid Res 17:517-522 (1989);Yoshikai et al. (1990); Yoshikai et al., Nucleic Acids Res 102:291-292(1991)). Yoshikai et al. (1990) describes the sequences of theexon-intron boundaries of the APP gene. These results taken togetherindicate that the miniimum size of the Alzheimer's gene is 175 kb. TABLE1 Alzheimer's Cosmid and Lambda Clones. Name of Insert Library CloneSize (kb) Assigned APP Region Cosmid 1 GPAPP47A 35 25 kb promoter & 9 kbintron 1 2 GPAAP36A 35 12 kb promoter & 22 kb intron 1 3 GAPP30A 30-355′ coding region 4 GAPP43A 30-35 exons 9, 10 and 11 Lambda 1 GAPP6A 12exon 6 2 GAPP6B 18 exons 4 and 5 3 GAPP20A 20 exon 6 4 GAPP20B 17 exons4 and 5 5 GAPP28A 18 exons 4 and 5 6 GAPP3A 14 exon 6 7 GAPP4A 19 exon 68 GAPP10A 16 exons 9, 10 and 11 9 GAPP16A 21 exon 6

[0063] Table 2 indicates where the 17 introns interrupt the APP codingsequence. The numbering refers to the nucleotide positions of APP770cDNA as shown in SEQ ID NO:5. The starting nucleotide of exon 1represents the first transcribed nucleotide. It is negative because the+1 nucleotide is the first nucleotide of the AUG initiator codon byconvention (Kang et al. (1988)). The ending nucleotide of exon 18represents the last nucleotide present in the mRNA prior to the poly(A)tail (Yoshikai et al. (1990)). It has been discovered that Yoshikai etal. (1990) and Yoshikai et al. (1991) contain an error in the locationof exon 8. FIG. 1 of Yoshikai et al. (1991) includes an EcoRI fragmentbetween EcoRI fragments containing exon 7 and exon 8. In fact, thisintervening EcoRI fragment is actually located immediately after exon 8,so that the EcoRI fragment containing exon 7 and the EcoRI fragmentcontaining exon 8 are adjacent to each other. TABLE 2 Location ofIntrons in APP Gene Sequence. Starting Ending Following nucleotidenucleotide Intron Exon 1 −146 57 Intron 1  Exon 2 58 225 Intron 2  Exon3 226 355 Intron 3  Exon 4 356 468 Intron 4  Exon 5 469 662 Intron 5 Exon 6 663 865 Intron 6  Exon 7 866 1033 Intron 7  Exon 8 1034 1090Intron 8  Exon 9 1091 1224 Intron 9  Exon 10 1225 1299 Intron 10 Exon 111300 1458 Intron 11 Exon 12 1459 1587 Intron 12 Exon 13 1588 1687 Intron13 Exon 14 1688 1909 Intron 14 Exon 15 1910 1963 Intron 15 Exon 16 19642064 Intron 16 Exon 17 2065 2211 Intron 17 Exon 18 2212 3432

[0064] APP Gene Mutations.

[0065] Certain families are genetically predisposed to Alzheimer'sdisease, a condition referred to as familial Alzheimer's disease (FAD),through mutations resulting in an amino acid replacement at position 717of the full length protein (Goate et al. (1991); Murrell et al. (1991);Chartier-Harlin et al. (1991)). These mutations co-segregate with thedisease within the families. For example, Murrell et al. (1991)described a specific mutation found in exon 17 (which Murrell et al.refers to as exon 15) where the valine of position 717 is replaced byphenylalanine.

[0066] Another FAD mutant form contains a change in amino acids atpositions 670 and 671 of the full length protein (Mullan et al. (1992)).In one form of this mutation, the lysine at position 670 is replaced byasparagine and the methionine at position 671 is replaced by leucine.The effect of this mutation is to increase the production of Aβ incultured cells approximately 7-fold (Citron et al., Nature 360: 672-674(1992); Lai et al., Science 259:514-516 (1993)). Replacement of themethionine at position 671 with leucine by itself has also been shown toincrease production of Aβ. Additional mutations in APP at amino acids669, 670, and 671 have been shown to reduce the amount of Aβ processedfrom APP (Citron et al., Neuron 14:661-670 (1995)). The APP constructwith Val at amino acid 690 produces an increased amount of a truncatedform of Aβ.

[0067] APP expression clones can be constructed that bear a mutation atamino acid 669, 670, 671, 690, 692, or 717 of the full length protein.The mutations from Lys to Asn and from Met to Leu at amino acids 670 and671, respectively, are sometimes referred to as the Swedish mutation.Additional mutations can also be introduced at amino acids 669, 670, or671 which either increase or reduce the amount of Aβ processed from APP.Mutations at these amino acids in any APP clone or transgene can becreated by site-directed mutagenesis (Vincent et al., Genes & Devel.3:334-347 (1989)), or, once made, can be incorporated into otherconstructs using standard genetic engineering techniques. Some mutationsat amino acid 717 are sometimes referred to as the Hardy mutation. Suchmutations can include conversion of the wild-type Val717 codon to acodon for Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, orGln. A preferred substitution for Val717 is Phe. These mutationspredispose individuals expressing the mutant proteins to developAlzheimer's disease. It is believed that the mutations affect theexpression and/or processing of APP, shifting the balance towardAlzheimer's pathology. Mutations at amino acid 669 can includeconversion of the wild-type Val669 codon to a codon for Trp, or deletionof the codon. Mutations at amino acid 670 can include conversion of thewild-type Lys670 codon to a codon for Asn or Glu, or deletion of thecodon. Mutations at amino acid 671 can include conversion of thewild-type Met671 codon to a codon for Leu, Val, Lys, Tyr, Glu, or Ile,or deletion of the codon. A preferred substitution for Lys670 is Asn,and a preferred substitution for Met671 is Leu. These mutationspredispose individuals expressing the mutant proteins to developAlzheimer's disease. The other listed mutations to amino acids 669, 670,and 671 are known to reduce the amount of Aβ processed from APP (Citronet al. (1995)). It is believed that these mutations affect processing ofAPP leading to a change in Aβ production.

[0068] Truncated forms of APP can also be expressed from transgeneconstructs. For example, APP cDNA truncated to encode amino acids 646 to770 of APP. The APP cDNA construct truncated to encode amino acids 646to 770 of APP, and operatively linked to the PDGF-B promoter, isreferred to as PDAPPc125.

[0069] Nucleic Acid Constructs Encoding Aβ-containing Proteins.

[0070] Constructs for use in transgenic animals include a promoter forexpression of the construct in a mammalian cell and a region encoding aprotein that includes all or a contiguous portion of one of the threeforms of APP: APP695, APP751, or APP770, with or without specific aminoacid mutations as described herein. It is preferred that protein encodedis an Aβ-containing protein. As used herein, an Aβ-containing protein isa protein that includes all or a contiguous portion of one of the threeforms of APP: APP695, APP751, or APP770, with or without specific aminoacid mutations as described herein, where the protein includes all or aportion of amino acids 672 to 714 of human APP. Preferred Aβ-containingproteins include amino acids 672 to 714 of human APP. Preferred forms ofsuch Aβ-containing proteins include all or a contiguous portion ofAPP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692,and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670,671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation inamino acid 669, 670, 671, 690, 692, and/or 717, where each of theseAβ-containing proteins includes amino acids 672 to 714 of human APP.

[0071] Preferred forms of the above Aβ-containing proteins are APP770;APP770 bearing a mutation in the codon encoding one or more amino acidsselected from the group consisting of amino acid 669, 670, 671, 690,692, 717; APP751; APP751 bearing a mutation in the codon encoding one ormore amino acids selected from the group consisting of amino acid 669,670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codonencoding one or more amino acids selected from the group consisting ofamino acid 669, 670, 671, 690, 692, 717; a protein consisting of aminoacids 646 to 770 of APP; a protein consisting of amino acids 670 to 770of APP; a protein consisting of amino acids 672 to 770 of APP; and aprotein consisting of amino acids 672 to 714 of APP.

[0072] In the constructs disclosed herein, the DNA encoding theAβ-containing protein can be cDNA or a cDNA/genomic DNA hybrid, whereinthe cDNA/genomic DNA hybrid includes at least one APP intron sequencewherein the intron sequence is sufficient for splicing.

[0073] Preferred constructs contain DNA encoding APP770; DNA encodingAPP770 bearing a mutation in the codon encoding amino acid 669, 670,671, 690, 692, 717, or a combination of these mutations; a fragment ofDNA encoding APP770 which encodes an amino acid sequence comprisingamino acids 672 to 714 of APP770; DNA encoding APP751; DNA encodingAPP751 bearing a mutation in the codon encoding amino acid 669, 670,671, 690, 692, 717, or a combination of these mutations; a fragment ofDNA encoding APP751 which encodes an amino acid sequence comprisingamino acids 672 to 714 of APP770; DNA encoding APP695; DNA encodingAPP695 bearing a mutation in the codon encoding amino acid 669, 670,671, 690, 692, 717, or a combination of these mutations; a fragment ofDNA encoding APP695 which encodes an amino acid sequence comprisingamino acids 672 to 714 of APP770; APP cDNA truncated to encode aminoacids 646 to 770 of APP; a combination cDNA/genomic DNA hybrid APP geneconstruct; a combination cDNA/genomic DNA hybrid APP gene constructbearing a mutation in the codon encoding amino acid 669, 670, 671, 690,692, 717, or a combination of these mutations; or a combinationcDNA/genomic DNA hybrid APP gene construct truncated at amino acid 671or 685.

[0074] Preferred forms of such constructs are APP770 cDNA; APP770 cDNAbearing a mutation in the codon encoding amino acid 669, 670, 671, 690,692, 717, or a combination of these mutations; a fragment of APP770 cDNAencoding an APP amino acid sequence, the amino acid sequence comprisingamino acids 672 to 714 of APP770; APP751 cDNA; APP751 cDNA bearing amutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717,or a combination of these mutations; a fragment of APP751 cDNA encodingan APP amino acid sequence, the amino acid sequence comprising aminoacids 672 to 714 of APP770; APP695 cDNA; APP695 cDNA bearing a mutationin the codon encoding amino acid 669, 670, 671, 690, 692, 717, or acombination of these mutations; a fragment of APP695 cDNA encoding anAPP amino acid sequence, the amino acid sequence comprising amino acids672 to 714 of APP770; APP cDNA truncated to encode amino acids 646 to770 of APP; a combination cDNA/genomic DNA hybrid APP gene construct; acombination cDNA/genomic DNA hybrid APP gene construct bearing amutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717,and a combination of these mutations; and a combination cDNA/genomic DNAhybrid APP gene construct truncated at amino acid 671 or 685.

[0075] Construction of Transgenes.

[0076] Construction of various APP transgenes can be accomplished usingany suitable genetic engineering technique, such as those described inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, N.Y., 1989). Regions of APP clones that have beenengineered or mutated can be interchanged by using convenientrestriction enzyme sites present in APP cDNA clones. A NruI site startsat position −5 (relative to the first nucleotide of the AUG initiatorcodon). A KpnI and an Asp718 site both start at position 57 (these areisoschizomers leaving different sticky ends). A XcmI site starts atposition 836 and cuts at position 843. A ScaI site starts at position1004. A XhoI site starts at position 1135. A BamHI site starts atposition 1554. A BglII site starts at position 1994. An EcoRI sitestarts at position 2020. A SpeI site starts at position 2583. AnotherEcoRI site starts at position 3076.

[0077] The clones bearing various portions of the human APP genesequence shown in FIGS. 1 to 5 can be constructed in a common mannerusing standard genetic engineering techniques. For example, these clonescan be constructed by first cloning the polyA addition signal from SV40virus, as a 253 base pair BclI to BamHI fragment (Reddy et al., Science200:494-502 (1978), into a modified vector from the pUC series. Next,the cDNA coding sequences (APP770, APP751, or APP695) can be inserted.Correct orientation and content of the fragments inserted can bedetermined through restriction endonuclease mapping and limitedsequencing. The clones bearing various carboxy terminal portions of thehuman APP gene sequence shown in FIGS. 4 and 5 can be constructedthrough several steps in addition to those indicated above. For example,an APP770 cDNA clone (SEQ ID NO:5) can be digested with Asp718 whichcleaves after nucleotide position 57. The resulting 5′ extension isfilled in using the Klenow enzyme (Sambrook et al. (1989)) and ligatedto a hexanucleotide of the following sequence: AGATCT, the recognitionsite for BglII. After cleavage with BglII, which also cuts afterposition 1994, and re-ligation, the translational reading frame of theprotein is preserved. The truncated protein thus encoded contains theleader sequence, followed by approximately 6 amino acids that precedethe Aβ, followed by the Aβ, and the 56 terminal amino acids of APP. Theclone in FIG. 5 is created by converting the nucleotide at position 2138to a T by site directed mutagenesis in the clone of FIG. 4a, thuscreating a termination codon directly following the last amino acidcodon of the Aβ. APP cDNA clones naturally contain an NruI site thatcuts 2 nucleotides upstream from the initiator methionine codon. Thissite can be used for attachment of the different promoters used tocomplete each construct.

[0078] APP transgenes can also be constructed using PCR cloningtechniques. Such techniques allow precise coupling of DNA fragments inthe transgenes.

[0079] Combination cDNA/Genomic DNA Clones.

[0080] Endogenous APP expression results from transcription of precursormRNA followed by alternative splicing to produce three main forms ofAPP. It is believed that this alternative splicing may be important inproducing the pattern of APP expression involved in Alzheimer's disease.It is also believed that the presence of introns in expressionconstructs can influence the level and nature of expression by, forexample, targeting precursor mRNA to mRNA processing and transportpathways (Huang et al., Nucleic Acids Res. 18:937-947 (1990)).Accordingly, transgenes combining cDNA and genomic DNA, which includeintron sequences, are a preferred type of construct.

[0081] The RNA splicing mechanism requires only a few specific and wellknown consensus sequences. Such sequences have been identified in APPgenomic DNA by Yoshikai et al. (1990). The disclosed transgenes can beconstructed using one or more complete and intact intron sequences.However, it is preferred that the transgenes are constructed usingtruncated intron sequences that contain an effective amount of intronsequence to allow splicing. In general, truncated intron sequences thatretain the splicing donor site, the splicing acceptor site, and thesplicing branchpoint sequence will constitute an effective amount of anintron. The sufficiency of any truncated intron sequence can bedetermined by testing for the presence of correctly spliced mRNA intransgenic cells using methods described below.

[0082] Other intron sequences and splicing signals which are not derivedfrom APP gene sequences may also be used in the transgene constructs.Such intron sequences will enhance expression of the transgeneconstruct. A preferred heterologous intron is a hybrid between theadenovirus major late region first exon and intron junction and an IgGvariable region splice acceptor. This hybrid intron can be constructed,for example, by joining the 162 bp PvuII to HindIII fragment of theadenovirus major late region, containing 8 bp of the first exon and 145bp of the first intron, and the 99 bp HindIII to PstI fragment of theIgG variable region splice acceptor clone-6, as described by Bothwell etal., Cell 24:625-637 (1981). A similar splice signal has been shown toenhance expression of a construct to which it was attached, as describedby Manley et al., Nucleic Acids Res. 18:937-947 (1990). It is preferredthat the heterologous intron be placed between the promoter and theregion encoding the APP.

[0083] A preferred APP combination cDNA/genomic expression cloneincludes an effective amount of introns 6, 7 and 8, as shown in FIG. 6.Such a transgene can be constructed as follows. A preferred method ofconstruction is described in Example 5. A plasmid containing the cDNAportion of the clone can be constructed by first converting the TaqIsite at position 860 in an APP770 cDNA clone to an XhoI site bysite-directed mutagenesis. Cleavage of the resulting plasmid with XhoIcuts at the new XhoI site and a pre-existing XhoI site at position 1135,and releases the KPI and OX-2 coding sequence. The plasmid thusgenerated serves as the acceptor for the KPI and OX-2 alternativesplicing cassette.

[0084] The alternative splicing cassette can be created through a seriesof cloning steps involving genomic DNA. First, the TaqI site at position860 in a genomic clone containing exon 6 and the adjacent downstreamintron can be converted to an XhoI site by site-directed mutagenesis.Cleavage of the resulting plasmid with XhoI cuts at the new XhoI siteand an XhoI site within either intron 6 or 7. This fragment, containinga part of exon 6 and at least a part of adjacent intron 6, can then becloned into the XhoI site in a plasmid vector. Second, a genomic clonecontaining exon 9 and the adjacent upstream genomic sequences is cleavedwith XhoI, cleaving the clone at the XhoI site at position 1135(position 910 using the numbering system of Kang et al. (1987)) and anXhoI site in either intron 7 or 8. This fragment, containing a part ofexon 9 and at least a part of adjacent intron 8, can then be cloned intothe XhoI site of another plasmid vector. These two exon/intron junctionfragments can then be released from their respective plasmid vectors bycleavage with XhoI and either BamHI or BglII, and cloned together intothe XhoI site of another plasmid vector. It is preferred that theexon/intron junction fragments be excised with BamHI. It is mostpreferable that BamHI sites are engineered in the intron portion of theexon/intron junction fragments prior to their excision. This allows theelimination of lengthy extraneous intron sequences from the cDNA/genomicclone.

[0085] The XhoI fragment resulting from cloning the two exon/intronjunction fragments together can be cleaved with either BamHI or BglII,depending on which enzyme was used for excision step above, and thegenomic 6.8 kb BamHI segment, containing the KPI and OX-2 coding regionalong with their flanking intron sequences, can be inserted. Thisfragment was identified by Kitaguchi et al. (1988) using Southern blotanalysis of BamHI-digested lymphocyte DNA from one normal individual andeight Alzheimer's disease patients using a 212 bp TaqI-AvaI fragment,nucleotides 862 to 1,073, of APP770 cDNA as the hybridization probe.Genomic DNA clones containing the region of the 225 bp insert can beisolated, for example, from a human leukocyte DNA library using the 212bp TaqI-AvaI fragment as a probe. In the genomic DNA, the 225 bpsequence is located in a 168 bp exon (exon 7) and a 57 bp exon (exon 8),separated by an intron of approximately 2.6 kb (intron 7), with bothexons flanked by intron-exon consensus sequences. The exon 7 correspondsto nucleotides 866 to 1,033 of APP770, and the exon 8 to nucleotides1,034 to 1,090. Exon 7 encodes the highly conserved region of theKunitz-type protease inhibitor family domain.

[0086] After cleavage with XhoI, this alternative splicing cassette,containing both exon and intron sequences, can then be excised bycleavage with XhoI and inserted into the XhoI site of the modifiedAPP770 cDNA plasmid (the acceptor plasmid) constructed above. Thesecloning steps generate a combination cDNA/genomic expression clone thatallows cells in a transgenic animal to regulate the inclusion of the KPIand OX-2 domains by a natural alternative splicing mechanism. Ananalogous gene bearing a mutation at amino acid 669, 670, 671, 690, 692,717, or a combination of these mutations, can be constructed eitherdirectly by in vitro mutagenesis. A mutation to amino acid 717 can alsobe made by using the mutated form of APP770 cDNA described above toconstruct an acceptor plasmid.

[0087] Promoters.

[0088] Different promoter sequences can be used to control expression ofnucleotide sequences encoding Aβ-containing proteins. The ability toregulate expression of the gene encoding an Aβ-containing protein intransgenic animals is believed to be useful in evaluating the roles ofthe different APP gene products in AD. The ability to regulateexpression of the gene encoding an Aβ-containing protein in culturedcells is believed to be useful in evaluating expression and processingof the different Aβ-containing gene products and may provide the basisfor cell cultured drug screens. A preferred promoter is the humanplatelet derived growth factor β (PDGF-B) chain gene promoter (Sasaharaet al., Cell 64:217-227 (1991)).

[0089] Preferred promoters for the disclosed APP constructs are thosethat, when operatively linked to the protein coding sequences, mediateexpression of one or more of the following expression products to atleast a specific level in brain tissue of a two to four month old animaltransgenic for one of the disclosed APP constructs. The products andtheir expression levels are Aβ_(tot) to a level of at least 30 ng/g (6.8pmoles/g) brain tissue and preferably at least 40 ng/g (9.12 pmoles/g)brain tissue, Aβ₁₋₄₂ to a level of at least 8.5 ng/g (1.82 pmoles/g)brain tissue and preferably at least 11.5 ng/g (2.5 pmoles/g) braintissue, full length APP (FLAPP) and APPα combined (FLAPP+APPα) to alevel of at least 150 pmoles/g brain tissue, APPα to a level of at least42 pmoles/g brain tissue, and mRNA encoding human Aβ-containing proteinto a level at least twice that of mRNA encoding the endogenous APP ofthe transgenic animal. Aβ_(tot) is the total of all forms of Aβ. Aβ₁₋₄₂is a form of Aβ having amino acids 1 to 42 of Aβ (corresponding to aminoacids 672 to 714 of APP). FLAPP+APPα refers to APP forms containing thefirst 12 amino acids of the Aβ region (corresponding to amino acids 672to 684 of APP). Thus, FLAPP+APPα represents a mixture of full lengthforms of APP and APP cleaved at the α-secretase site (Esch et al.,Science 248:1122-1124 (1990)). APPβ is APP cleaved at the β-secretasesite (Seubert et al., Nature 361:260-263 (1993)).

[0090] It is intended that the levels of expression described aboverefer to amounts of expression product present and are not limited tothe specific units of measure used above. Thus, an expression level canbe measured, for example, in moles per gram of tissue, grams per gramsof tissue, moles per volume of tissue, and in grams per volume oftissue. The equivalence of these units of measure to the measures listedabove can be determined using known conversion methods.

[0091] The levels of expression described above need not occur in allbrain tissues. Thus, a promoter is considered preferred if at least oneof the levels of expression described above occurs in at least one typeof brain tissue. Where expression is tissue-specific, it is understoodthat if the expression level is sufficient in the specific brain tissue,the promoter is considered preferred even though the expression level inbrain tissue as a whole may not, and need not, reach a threshold level.It is preferred that this level of expression is observed in hippocampaland/or cortical brain tissue. The promoter can mediate expression of theabove expression products to the levels described above eitherconstitutively or by induction. Induction can be accomplished by, forexample, administration of an activator molecule, by heat, or byexpression of a protein activator of transcription for the promoteroperatively linked to the gene encoding an Aβ-containing protein. Manyinducible expression systems which would be suitable for this purposeare known to those of skill in the art.

[0092] It is preferred that, in making the above measurements, the braintissue is prepared by the following method. A brain from a transgenictest animal is dissected and the tissue is kept on ice throughout thehomogenization procedure except as noted. The brain tissue ishomogenized in 10 volumes (w/v) of 5 M guanidine-HCl, 50 mM Tris-HCl, pH8.5. The sample is then gently mixed for 2 to 4 hours at roomtemperature. Homogenates are then diluted 1:10 in cold casein buffer #1(0.25% casein/phosphate buffered saline (PBS) 0.05% sodium azide, pH7.4, 1× protease inhibitor cocktail) for a final 0.5 M guanidineconcentration and kept on ice. 100× protease inhibitor cocktail iscomposed of 2 mg/ml aprotinin, 0.5 M EDTA, pH 8.0, 1 mg/ml leupeptin.Diluted homogenates are then spun in an Eppendorf microfuge at 14,000rpm for 20 minutes at 4° C. If further dilutions are required, they canbe made with cold guanidine buffer #2 (1 part guanidine buffer #1 to 9parts casein buffer #1).

[0093] It is preferred that the following assay be used to identifypreferred promoters for their ability to mediate expression of Aβ to thelevels described above. Antibody 266 (Seubert et al., Nature 359:325-327(1992)) is dissolved at 10 μg/ml in buffer (0.23 g/L NaH₂PO₄-H₂O, 26.2g/L NaHPO₄-7H₂O, 1 g/L sodium azide adjusted to pH 7.4) and 100 μl/wellis coated onto 96-well immunoassay plates (Costar) and allowed to bindovernight. The plate is then aspirated and blocked for at least 1 hourwith a 0.25% human serum albumin solution in 25 g/L sucrose, 10.8 g/LNa₂HPO₄-7H₂O, 1.0 g/L NaH₂PO₄-H₂O, 0.5 g/L sodium azide adjusted to pH7.4. The 266 coated plate is then washed 1× with wash buffer (PBS/0.05%Tween 20) using a Skatron plate washer. 100 μl/well of Aβ1-40 standardsand brain tissue samples are added to the plate in triplicate andincubated overnight at 4° C. Aβ1-40 standards are made from 0.0156,0.0312, 0.0625, 0.125, 0.250, 0.500, and 1.000 μg/ml stocks in DMSOstored at −40° C. as well as a DMSO only control for backgrounddetermination. Aβ standards consist of 1:100 dilution of each standardinto guanidine buffer #3 (1 part BSA buffer to 9 parts guanidine buffer#1) followed by a 1:10 dilution into casein buffer #1 (Note: the finalAβ concentration range is 15.6 to 1000 pg/ml and the final guanidineconcentration is 0.5 M). BSA buffer consists of 1% bovine serum albumin(BSA, immunoglobulin-free)/PBS/0.05% sodium azide. The plates and caseinbuffer #2 (0.25% casein/PBS/0.05% Tween 20/pH 7.4) are then brought toroom temperature (RT). The plates are then washed 3× with wash buffer.Next, 100 μl/well of 3D6-biotin at 0.5 μg/ml in casein buffer #2 isadded to each well and incubated at 1 hour at RT.

[0094] Monoclonal antibody 3D6 was raised against the synthetic peptideDAEFRGGC (SEQ ID NO:10) which was conjugated through the cysteine tosheep anti-mouse immunoglobulin. The antibody does not recognizesecreted APP but does recognize species that begin at Aβ position 1(Asp). For biotinylating 3D6, follow Pierce's NHS-Biotin protocol forlabeling IgG (cat. #20217X) except use 100 mM sodium bicarbonate, pH 8.5and 24 mg NHS-biotin per ml of DMSO.

[0095] The plates are then again washed 3× with wash buffer. Then, 100μl/well of horseradish peroxidase (HRP)-avidin (Vector Labs, cat. #A-2004) diluted 1:4000 in casein buffer #2 is added to each well andincubated for 1 hour at RT. The plates are washed 4× with wash bufferand then 100 μl/well of TMB substrate (Slow TMB-ELISA (Pierce cat. #34024)) at RT is added to each well and incubated for 15 minutes at RT.Finally, 25 μl/well of 2 N H₂SO₄ is added to each well to stop theenzymatic reaction, and the plate is read at 450 nm to 650 nm using theMolecular Devices Vmax reader.

[0096] It is preferred that the relative levels of mRNA encoding humanAβ-containing protein mRNA encoding the endogenous APP of the transgenicanimal be measured in the manner described by Bordonaro et al.,Biotechniques 16:428-430 (1994), and Rockenstein et al., J. Biol. Chem.270:28257-28267 (1995). Preferred methods for measuring the expressionlevel of Aβ₁₋₄₂, FLAPP+APPα, and APPβ are described in Example 8.

[0097] Yeast Artificial Chromosomes.

[0098] The constructs shown in FIG. 7 can be constructed as follows.Large segments of human genomic DNA, when cloned into certain vectors,can be propagated as autonomously-replicating units in the yeast cell.Such vector-borne segments are referred to as yeast artificialchromosomes (YAC; Burke et al. Science 236:806 (1987)). A human YAClibrary is commercially available (Clontech, Palo Alto, Calif.) with anaverage insert size of 250,000 base pairs (range of 180,000 to 500,000base pairs). A YAC clone of the Alzheimer's gene can be directlyisolated by screening the library with the human APP770 cDNA. Theinclusion of all of the essential gene regions in the clone can beconfirmed by PCR analysis.

[0099] The YAC-APP clone, shown in FIG. 7a, can be established inembryonic stem (ES) cells by selecting for neomycin resistance encodedby the YAC vector. ES cells bearing the YAC-APP clone can be used toproduce transgenic mice by established methods described below under“Transgenic Mice” and “Embryonic Stem Cell Methods”. The YAC-APP genebearing a mutation at amino acid 717 (FIG. 7b) can be produced throughthe generation of a YAC library using genomic DNA from a person affectedby a mutation at amino acid 717. Such a clone can be identified andestablished in ES cells as described above.

[0100] Genetic Alteration of the Mouse APP Gene.

[0101] The nucleotide sequence homology between the human and murineAlzheimer's protein genes is approximately 85%. Within the Aβ-codingregion, there are three amino acid differences between the twosequences. Amino acids Lys 670, Met671, and Val717,which can be mutatedto alter APP processing, are conserved between mouse, rat, and man.Wild-type rodents do not develop Alzheimer's disease nor do they developdeposits or plaques in their central nervous system (CNS) analogous tothose present in human Alzheimer's patients. Therefore, it is possiblethat the human but not the rodent form of Aβ is capable of causingdisease. Homologous recombination (Capecchi, Science 244:1288-1292(1989)) can be used to convert the mouse Alzheiner's gene in situ to agene encoding the human Aβ by gene replacement. This recombination isdirected to a site downstream from the KPI and OX-2 domains, forexample, within exon 9, so that the natural alternative splicingmechanisms appropriate to all cells within the transgenic animal can beemployed in expressing the final gene product.

[0102] Both wild-type (FIG. 8a) and mutant (FIG. 8b) forms of human cDNAcan be used to produce transgenic models expressing either the wild-typeor mutant forms of APP. The recombination vector can be constructed froma human APP cDNA (APP695, APP751, or APP770 form), either wild-type,mutant at amino acid 669, 670, 671, 690, 692, 717, or a combination ofthese mutations. Cleavage of the recombination vector, for example, atthe XhoI site within exon 9, promotes homologous recombination withinthe directly adjacent sequences (Capecchi (1989)). The endogenous APPgene resulting from this event would be normal up to the point ofrecombination, within exon 9 in this example, and would consist of thehuman cDNA sequence thereafter.

[0103] Preparation of Constructs for Transfections and Microinjections.

[0104] DNA clones for microinjection are cleaved with enzymesappropriate for removing the bacterial plasmid sequences, such as SalIand NotI, and the DNA fragments electrophoresed on 1% agarose gels inTBE buffer (Sambrook et al. (1989)). The DNA bands are visualized bystaining with ethidium bromide, and the band containing the APPexpression sequences is excised. The excised band is then placed indialysis bags containing 0.3 M sodium acetate, pH 7.0. DNA iselectroeluted into the dialysis bags, extracted with phenol-chloroform(1:1), and precipitated by two volumes of ethanol. The DNA isredissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4,and 1 mM EDTA) and purified on an Elutip-D™ column. The column is firstprimed with 3 ml of high salt buffer (1 M NaCl, 20 mM Tris, pH 7.4, and1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNAsolutions are passed through the column for three times to bind DNA tothe column matrix. After one wash with 3 ml of low salt buffer, the DNAis eluted with 0.4 ml of high salt buffer and precipitated by twovolumes of ethanol. DNA concentrations are measured by absorption at 260nm in a UV spectrophotometer. For microinjection, DNA concentrations areadjusted to 3 μg/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other methodsfor purification of DNA for microinjection are also described in Hoganet al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1986); in Palmiter et al., Nature 300:611(1982); in The Qiagenologist, Application Protocols, 3rd edition,published by Qiagen, Inc., Chatsworth, Calif.; and in Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989).

[0105] Construction of Transgenic Animals.

[0106] A. Animal Sources.

[0107] Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Charles River (Wilmington, Mass.),Taconic (Germantown, N.Y.), and Harlan Sprague Dawley (Indianapolis,Ind.). Many strains are suitable, but Swiss Webster (Taconic) femalemice are preferred for embryo retrieval and transfer. B6D2F₁ (Taconic)males can be used for mating and vasectomized Swiss Webster studs can beused to stimulate pseudopregnancy. Vasectomized mice and rats can beobtained from the supplier.

[0108] B. Microinjection Procedures.

[0109] The procedures for manipulation of the rodent embryo and formicroinjection of DNA are described in detail in Hogan et al.,Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1986), the teachings of which are incorporatedherein.

[0110] C. Transgenic Mice.

[0111] Female mice six weeks of age are induced to superovulate with a 5IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG;Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of humanchorionic gonadotropin (hCG; Sigma). Females are placed with malesimmediately after hCG injection. Twenty-one hours after hCG injection,the mated females are sacrificed by CO₂ asphyxiation or cervicaldislocation and embryos are recovered from excised oviducts and placedin Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin(BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase(1 mg/ml). Pronuclear embryos are then washed and placed in Earle'sbalanced salt solution containing 0.5% BSA (EBSS) in a 37.5° C.incubator with a humidified atmosphere at 5% CO₂, 95% air until the timeof injection. Embryos can be implanted at the two cell stage.

[0112] Randomly cycling adult female mice are paired with vasectomizedmales. Swiss Webster or other comparable strains can be used for thispurpose. Recipient females are mated at the same time as donor females.At the time of embryo transfer, the recipient females are anesthetizedwith an intraperitoneal injection of 0.015 ml of 2.5% avertin per gramof body weight. The oviducts are exposed by a single midline dorsalincision. An incision is then made through the body wall directly overthe oviduct. The ovarian bursa is then torn with watchmakers forceps.Embryos to be transferred are placed in DPBS (Dulbecco's phosphatebuffered saline) and in the tip of a transfer pipet (about 10 to 12embryos). The pipet tip is inserted into the infundibulum and theembryos transferred. After the transfer, the incision is closed by twosutures.

[0113] D. Transgenic Rats.

[0114] The procedure for generating transgenic rats is similar to thatof mice (Hammer et al., Cell 63:1099-112 (1990)). Thirty day-old femalerats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48hours later each female placed with a proven male. At the same time,40-80 day old females are placed in cages with vasectomized males. Thesewill provide the foster mothers for embryo transfer. The next morningfemales are checked for vaginal plugs. Females who have mated withvasectomized males are held aside until the time of transfer. Donorfemales that have mated are sacrificed (CO₂ asphyxiation) and theiroviducts removed, placed in DPBS (Dulbecco's phosphate buffered saline)with 0.5% BSA and the embryos collected. Cumulus cells surrounding theembryos are removed with hyaluronidase (1 mg/ml). The embryos are thenwashed and placed in EBSS (Earle's balanced salt solution) containing0.5% BSA in a 37.5° C. incubator until the time of microinjection.

[0115] Once the embryos are injected, the live embryos are moved to DPBSfor transfer into foster mothers. The foster mothers are anesthetizedwith ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip). A dorsalmidline incision is made through the skin and the ovary and oviduct areexposed by an incision through the muscle layer directly over the ovary.The ovarian bursa is torn, the embryos are picked up into the transferpipet, and the tip of the transfer pipet is inserted into theinfundibulum. Approximately 10 to 12 embryos are transferred into eachrat oviduct through the infundibulum. The incision is then closed withsutures, and the foster mothers are housed singly.

[0116] E. Embryonic Stem (ES) Cell Methods.

[0117] 1. Introduction of cDNA into ES Cells.

[0118] Methods for the culturing of ES cells and the subsequentproduction of transgenic animals, the introduction of DNA into ES cellsby a variety of methods such as electroporation, calcium phosphate/DNAprecipitation, and direct injection are described in detail inTeratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), the teachings of which are incorporatedherein. Selection of the desired clone of transgene-containing ES cellscan be accomplished through one of several means. For random geneintegration, an APP clone is co-precipitated with a gene encodingneomycin resistance. Transfection is carried out by one of severalmethods described in detail in Lovell-Badge, in Teratocarcinomas andEmbryonic Stem Cells, A Practical Approach, ed. E. J. Robertson, (IRLPress 1987), or in Potter et al., Proc. Natl. Acad. Sci. USA 81:7161(1984). Lipofection can be performed using reagents such as provided incommercially available kits, for example DOTAP (Boehringer-Mannheim) orlipofectin (BRL). Calcium phosphate/DNA precipitation, lipofection,direct injection, and electroporation are the preferred methods. Inthese procedures, 0.5×10⁶ ES cells are plated into tissue culture dishesand transfected with a mixture of the linearized APP clone and 1 mg ofpSV2neo DNA (Southern and Berg, J. Mol. Appl. Gen. 1:327-341 (1982))precipitated in the presence of 50 mg lipofectin (BRL) in a final volumeof 100 μl. The cells are fed with selection medium containing 10% fetalbovine serum in DMEM supplemented with G418 (between 200 and 500 μg/ml).Colonies of cells resistant to G418 are isolated using cloning rings andexpanded. DNA is extracted from drug resistant clones and Southern blotsusing an APP770 cDNA probe can be used to identify those clones carryingthe APP sequences. PCR detection methods may also used to identify theclones of interest.

[0119] DNA molecules introduced into ES cells can also be integratedinto the chromosome through the process of homologous recombination,described by Capecchi (1989). Direct injection results in a highefficiency of integration. Desired clones can be identified through PCRof DNA prepared from pools of injected ES cells. Positive cells withinthe pools can be identified by PCR subsequent to cell cloning (Zimmerand Gruss, Nature 338:150-153 (1989). DNA introduction byelectroporation is less efficient and requires a selection step. Methodsfor positive selection of the recombination event (for example, neoresistance) and dual positive-negative selection (for example, neoresistance and gancyclovir resistance) and the subsequent identificationof the desired clones by PCR have been described by Joyner et al.,Nature 338:153-156 (1989), and Capecchi (1989), the teachings of whichare incorporated herein.

[0120] 2. Embryo Recovery and ES Cell Injection.

[0121] Naturally cycling or superovulated female mice mated with malescan be used to harvest embryos for the implantation of ES cells. It isdesirable to use the C57BL/6 strain for this purpose when using mice.Embryos of the appropriate age are recovered approximately 3.5 daysafter successful mating. Mated females are sacrificed by CO₂asphyxiation or cervical dislocation and embryos are flushed fromexcised uterine horns and placed in Dulbecco's modified essential mediumplus 10% calf serum for injection with ES cells. Approximately 10 to 20ES cells are injected into blastocysts using a glass microneedle with aninternal diameter of approximately 20 μm.

[0122] 3. Transfer of Embryos to Pseudopregnant Females.

[0123] Randomly cycling adult female mice are paired with vasectomizedmales. Mouse strains such as Swiss Webster, ICR or others can be usedfor this purpose. Recipient females are mated such that they will be at2.5 to 3.5 days post-mating when required for implantation withblastocysts containing ES cells. At the time of embryo transfer, therecipient females are anesthetized with an intraperitoneal injection of0.015 ml of 2.5% avertin per gram of body weight. The ovaries areexposed by making an incision in the body wall directly over the oviductand the ovary and uterus are externalized. A hole is made in the uterinehorn with a 25 gauge needle through which the blastocysts aretransferred. After the transfer, the ovary and uterus are pushed backinto the body and the incision is closed by two sutures. This procedureis repeated on the opposite side if additional transfers are to be made.

[0124] Identification, Characterization, and Utilization of TransgenicMice and Rats.

[0125] Transgenic rodents can be identified by analyzing their DNA. Forthis purpose, tail samples (1 to 2 cm) can be removed from three weekold animals. DNA from these or other samples can then be prepared andanalyzed by Southern blot, PCR, or slot blot to detect transgenicfounder (F₀) animals and their progeny (F₁ and F₂).

[0126] A. Pathological Studies.

[0127] The various F₀, F₁, and F₂ animals that carry a transgene can beanalyzed by immunohistology for evidence of Aβ deposition, expression ofAPP or APP cleavage products, neuronal or neuritic abnormalities, andinflammatory responses in the brain. Brains of mice and rats from eachtransgenic line are fixed and then sectioned. Sections are stained withantibodies reactive with the APP and/or the Aβ. Secondary antibodiesconjugated with fluorescein, rhodamine, horse radish peroxidase, oralkaline phosphatase are used to detect the primary antibody. Thesemethods permit identification of amyloid plaques and other pathologicallesions in specific areas of the brain. Plaques ranging in size from 9to >50 μm characteristically occur in the brains of AD patients in thecerebral cortex, but also may be observed in deeper grey matterincluding the amygdaloid nucleus, corpus striatum and diencephalon.Sections can also be stained with other antibodies diagnostic ofAlzheimer's plaques, recognizing antigens such as APP, Alz-50, tau,A2B5, neurofilaments, synaptophysin, MAP-2, ubiquitin, complement,neuron-specific enolase, and others that are characteristic ofAlzheimer's pathology (Wolozin et al., Science 232:648 (1986); Hardy andAllsop, Trends in Pharm. Sci. 12:383-388 (1991); Selkoe, Ann. Rev.Neurosci. 12:463-490 (1989); Arai et al., Proc. Natl. Acad. Sci. USA87:2249-2253 (1990); Majocha et al., Amer. Assoc. Neuropathology Abs99:22 (1988); Masters et al., Proc. Natl. Acad. Sci. 82:4245-4249(1985); Majocha et al., Can J Biochem Cell Biol 63:577-584 (1985)).Staining with thioflavin S and Congo Red can also be carried out toanalyze the presence of amyloid and co-localization of Aβ depositswithin neuritic plaques and NFTs.

[0128] B. Analysis of APP and Aβ Expression.

[0129] 1. mRNA.

[0130] Messenger RNA can be isolated by the acid guanidiniumthiocyanate-phenol:chloroform extraction method (Chomaczynski andSacchi, Anal Biochem 162:156-159 (1987)) from cell lines and tissues oftransgenic animals to determine expression levels by Northern blots,RNAse and nuclease protection assays.

[0131] 2. Protein.

[0132] APP, Aβ, and other fragments of APP can and have been detected byusing polyclonal and monoclonal antibodies that are specific to the APPextra-cytoplasmic domain, Aβ region, Aβ₁₋₄₂, Aβ₁₋₄₀, APPβ, FLAPP+APPα,and C-terminus of APP. A variety of antibodies that are human sequencespecific, such as 10D5 and 6C6, are very useful for this purpose (Gameset al. (1995)).

[0133] 3. Western Blot Analysis.

[0134] Protein fractions can be isolated from tissue homogenates andcell lysates and subjected to Western blot analysis as described by, forexample, Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor, N.Y., 1988); Brown et al., J. Neurochem. 40:299-308 (1983); andTate-Ostroff et al., Proc Natl Acad Sci 86:745-749 (1989).

[0135] Briefly, the protein fractions are denatured in Laemmli samplebuffer and electrophoresed on SDS-Polyacrylamide gels. The proteins arethen transferred to nitrocellulose filters by electroblotting. Thefilters are blocked, incubated with primary antibodies, and finallyreacted with enzyme conjugated secondary antibodies. Subsequentincubation with the appropriate chromogenic substrate reveals theposition of APP derived proteins.

[0136] C. Pathological and Behavioral Studies.

[0137] 1. Pathological Studies.

[0138] Immunohistology and thioflavin S staining are conducted asdescribed elsewhere herein.

[0139] In situ Hybridizations:

[0140] Radioactive or enzymatically labeled nucleic acid probes can beused to detect mRNA in situ. The probes are degraded or prepared to beapproximately 100 nucleotides in length for better penetration of cells.The hybridization procedure of Chou et al., J. Psych. Res. 24:27-50(1990), for fixed and paraffin embedded samples is briefly describedbelow although similar procedures can be employed with samples sectionedas frozen material. Paraffin slides for in situ hybridization aredewaxed in xylene and rehydrated in a graded series of ethanols andfinally rinsed in phosphate buffered saline (PBS). The sections arepost-fixed in fresh 4% paraformaldehyde. The slides are washed with PBStwice for 5 minutes to remove paraformaldehyde. Then the sections arepermeabilized by treatment with a 20 μg/ml proteinase K solution. Thesections are re-fixed in 4% paraformaldehyde, and basic molecules thatcould give rise to background probe binding are acetylated in a 0.1 Mtriethanolamine, 0.3 M acetic anhydride solution for 10 minutes. Theslides are washed in PBS, then dehydrated in a graded series of ethanolsand air dried. Sections are hybridized with antisense probe, using senseprobe as a control. After appropriate washing, bound radioactive probesare detected by autoradiography or enzymatically labeled probes aredetected through reaction with the appropriate chromogenic substrates.

[0141] 2. Behavioral Studies.

[0142] Behavioral tests designed to assess learning and memory deficitsare employed. An example of such as test is the Morris water maze(Morris, Learn Motivat. 12:239-260 (1981)). In this procedure, theanimal is placed in a circular pool filled with water, with an escapeplatform submerged just below the surface of the water. A visible markeris placed on the platform so that the animal can find it by navigatingtoward a proximal visual cue. Alternatively, a more complex form of thetest in which there are no local cues to mark the platform's locationwill be given to the animals. In this form, the animal must learn theplatform's location relative to distal visual cues, and can be used toassess both reference and working memory. A learning deficit in thewater maze has been demonstrated with PDAPP transgenic mice. An exampleof behavioral analysis for assessing the effect of transgenic expressionof Aβ-containing proteins is described in Example 9.

[0143] Operant Behavior Studies of Memory Function:

[0144] Memory function of the disclosed transgenic animals can beassessed by testing memory-related feeding behavior (Dunnett, “Operantdelayed matching and non-matching position in rats” in BehavioralNeuroscience, Volume I: A Practical Approach (Sagal, ed., IRL Press,N.Y., 1993) pages 123-136; Zornetzen, Behav. Neur. Biol. 36:49-60(1982)). Transgenic and non-transgenic mice, are trained to earn foodrewards in a two component operant procedure. One component features adelayed spatial alternation schedule. Under this schedule, the mousemust remember over a variable time delay which lever it has pressed inthe previous trial so that it can earn a reward by pressing thealternate lever on the current trial. This provides a measure of theanimal's recent or “working” memory. The second component features adiscrimination spatial alternation schedule. Under this schedule, themouse earns a reward by pressing whatever lever is illuminated. Thisdiscrimination behavior is an example of reference memory. These twogroups of mice, transgenic and non-transgenic, can be chronicallystudied over time, for example, from 3 months of age until the end oftheir useful life span, in order to assess the development ofsensitivity to cholinergic antagonists and behavioral impairment onthese memory tasks. It is expected that the disclosed transgenic micewill model the cognitive deficits of Alzheimer's disease with enhancedsensitivity to the memory-disrupting effects of cholinergic antagonistsand impairment on “working” and reference memory tasks. Dose-responsechallenges with the cholinergic antagonist can be conducted at variousages. These memory behavioral tests can also be used to compare theeffect of compounds on the behavioral impairment of the disclosedtransgenic animals. In this case, the two groups of mice are transgenicmice to which a test compound is administered and transgenic mice towhich the compound is not administered.

[0145] Emotional Reactivity and Object Recognition:

[0146] Various functions of the disclosed transgenic animals can beassessed by testing locomotor activity, emotional reactivity to a novelenvironment or to novel objects, and object recognition. A first set ofassessments are performed in the same animals at different ages (eachanimal is its own control) in order to test their performance in termsof locomotor activity, emotional reactivity to a novel environment or tonovel objects, and object recognition, a form of memory which isseverely impaired in AD patients. On the first day, transgenic andnon-transgenic control mice are individually placed in a square openfield with a central platform. For 30 minutes, horizontal and verticalactivity, and crossings of the platform, are recorded by blocks of 5minutes for each animal. On the second day, each animal is submitted totwo trials with an intertrial of 1 hour. On the first trial, twoidentical objects are placed in the open field and the animal is allowed3 minutes of exploration. On the second trial, one of the objects isreplaced by a new object and the time spent by the animal in exploringthe familiar and novel object is recorded during the next 3 minutes(Ennaceur and Delacour, Behav. Brain Res. 31:47-59 (1988)). Animals arethen tested for neophobic behavior, which is considered as an index ofanxiety, in a free exploration situation, in which animals are given theopportunity to move freely between a familiar and a novel environment.

[0147] Thereafter, the same animals are submitted to various learningtasks to investigate their learning and memory capacities. They arefirst tested for spatial recognition memory in a T-maze delayedalternation task at 6 hour and 24 hour delays. This form of memory hasbeen shown to be very sensitive to hippocampal damage. One half of theanimals of each group is then trained in a positively reinforcedlever-press task as described above. This can be used to measure posttraining improvement in performance of the animals, which has been shownto involve hippocampal activation. The other half of the animals istrained in spatial discrimination in an 8 arm radial maze (Oltons andSamuelson, J. Exp. Psychol. [Animal Behav.] 2:97-116 (1976)) in order toevaluate working and reference memory and to analyze their strategies(angle preference), which give a better index of memory capacities. Theanimals trained and tested in the bar-lever press task at 2 to 3 monthsold can be trained and tested in radial maze at 9 to 10 months old, andvice-versa. A working memory deficit has been demonstrated in PDAPPtransgenic mice in the radial arm maze.

[0148] Two additional groups can also be submitted to the samebehavioral tests as above at 9 to 10 months old in order to determinewhether behavioral screening performed at 2 to 3 months old influencedfurther learning and memory capacities.

[0149] These memory behavioral tests can also be used to compare theeffect of compounds on the behavioral impairment of the disclosedtransgenic animals. In this case, the two groups of mice are transgenicmice to which a test compound is administered, and transgenic mice towhich the compound is not administered.

[0150] The procedures applied to test transgenic mice are similar fortransgenic rats.

[0151] D. Preferred Characteristics.

[0152] The above phenotypic characteristics of the disclosed transgenicanimals can be used to identify those forms of the disclosed transgenicanimals that are preferred as animal models. Additional phenotypiccharacteristics, and assays for measuring these characteristics, thatcan also be used to identify those forms of the disclosed transgenicanimals that are preferred as animal models, are described in Example 6.These characteristics are preferably those that are similar tophenotypic characteristics observed in Alzheimer's disease. APP and Aβmarkers which are also useful for identifying those forms of thedisclosed transgenic animals that are preferred as animal models aredescribed below. Any or all of the these markers or phenotypiccharacteristics can be used either alone or in combination to identifypreferred forms of the disclosed transgenic animals. For example, thepresence of plaques in brain tissue that can be stained with Congo redis a phenotypic characteristic which can identify a disclosed transgenicanimal as preferred. It is intended that the levels of expression ofcertain APP-related proteins present in preferred transgenic animals(discussed above) is an independent characteristic for identifyingpreferred transgenic animals. Thus, the most preferred transgenicanimals will exhibit both a disclosed expression level for one or moreof the APP-related proteins and one or more of the phenotypiccharacteristics discussed above. Especially preferred phenotypiccharacteristics (the presence of which identifies the animal as apreferred transgenic animal) are the presence of amyloid plaques thatcan be stained with Congo Red (Kelly (1984)), the presence ofextracellular amyloid fibrils as identified by electron microscopy by 12months of age, and the presence of type I dystrophic neurites asidentified by electron microscopy by 12 months of age (composed ofspherical neurites that contain synaptic proteins and APP; Dickson etal., Am J Pathol 132:86-101 (1988); Dickson et al., Acta Neuropath.79:486-493 (1990); Masliah et al., J Neuropathol Exp Neurol 52:135-142(1993); Masliah et al., Acta Neuropathol 87:135-142 (1994); Wang andMunoz, J Neuropathol Exp Neurol 54:548-556 (1995)). Examples of thedetection of these characteristics is provided in Example 6. It is mostpreferred that the transgenic animals have amyloid plaques that can bestained with Congo Red as of 14 months of age.

[0153] Screening of Compounds for Treatment of Alzheimer's Disease.

[0154] The transgenic animals, or animal cells derived from transgenicanimals, can be used to screen compounds for a potential effect in thetreatment of Alzheimer's disease using standard methodology. In such ADscreening assays, the compound is administered to the animals, orintroduced into the culture media of cells derived from these animals,over a period of time and in various dosages, then the animals or animalcells are examined for alterations in APP expression or processing,expression levels or localization of other AD markers, histopathology,and/or, in the case of animals, behavior using the procedures describedabove and in the examples below. In general, any improvement inbehavioral tests, alteration in AD-associated markers, reduction in theseverity of AD-related histopathology, reduction in the expression of Aβor APP cleavage products, and/or changes in the presence, absence orlevels of other compounds that are correlated with AD which are observedin treated animals, relative to untreated animals, is indicative of acompound useful for treating Alzheimer's disease. The specific proteins,and the encoding transcripts, the enzymatic or biochemical activity,and/or histopathology of those proteins, that are associated with andcharacteristic of AD are referred to herein as markers. Expression orlocalization of these markers characteristic of AD has either beendetected, or is expected to be present, in the disclosed transgenicanimals. These markers can be measured or detected, and thosemeasurements compared between treated and untreated transgenic animalsto determine the effect of a tested compound.

[0155] Markers useful for AD screening assays are selected based ondetectable changes in these markers that are associated with AD. Manysuch markers have been identified in AD and have either been detected inthe disclosed transgenic animals or are expected to be present in theseanimals. These markers fall into several categories based on theirnature, location, or function. Preferred examples of markers useful inAD screening assays are described below, group as Aβ-related markers,plaque-related markers, cytoskeletal and neuritic markers, inflammatorymarkers, and neuronal and neurotransmitter-related markers.

[0156] A. Aβ-related Markers.

[0157] Expression of the various forms of APP and Aβ can be directlymeasured and compared in treated and untreated transgenic animals bothby immunohistochemistry and by quantitative ELISA measurements asdescribed above and in the examples. Currently, it is known that twoforms of APP products are found, APP and Aβ (Haass and Selkoe, Cell75:1039-1042 (1993)). They have been shown to be intrinsicallyassociated with the pathology of AD in a time dependent manner.Therefore, preferred assays compare age-related changes in APP and Aβexpression in the transgenic mice. As described in Example 6, increasesin Aβ have been demonstrated during aging of the PDAPP mouse.

[0158] Preferred targets for assay measurement are Aβ markers known toincrease in individuals with Alzheimer's disease are total Aβ (Aβ_(tot))Aβ 1-42 (Aβ₁₋₄₂; Aβ with amino acids 1-42), Aβ₁₋₄₀ (Aβ with amino acids1-40), Aβ N3(pE) (Aβ_(N3)(pE)); Aβ_(X-42) (Aβ_(X-42); Aβ forms ending atamino acid 42); Aβ X-40 (Aβ_(X-40); Aβ forms ending at amino acid 40);insoluble Aβ (Aβ_(Isoluble)); and soluble Aβ (Aβ_(Soluble); Kuo et al.,J. Biol. Chem. 271(8):4077-4081 (1996)). Aβ_(N3)(pE) has pyroglutamicacid at position 3 (Saido, Neuron 14:457-466 (1995)). Aβ_(X-42) refersto any of the C-terminal forms of Aβ such as Aβ₁₃₋₄₂. Aβ_(Insoluble)refers to forms of Aβ that are recovered as described in Gravina, J.Biol. Chem. 270:7013-7016 (1995). APPβ can also be specifically measuredto assess the amount of β-secretase activity (Seubert et al., Nature361:260-263 (1993)). Several of these Aβ forms and their associationwith Alzheimer's disease are described by Haass and Selkoe (1993).Detection and measurement of Aβ_(tot), Aβ₁₋₄₂, and Aβ_(X-42) aredescribed in Example 6. Generally, specific forms of Aβ can be assayed,either quantitatively or qualitatively using specific antibodies, asdescribed below. When referring to amino acid positions in forms of Aβ,the positions correspond to the Aβ region of APP. Amino acid 1 of Aβcorresponds to amino acid 672 of APP, and amino acid 42 of Aβcorresponds to amino acid 714 of APP.

[0159] Also preferred as targets for assay measurement are APP markers.For example, different forms of secreted APP (termed APPα and APPβ) canalso be measured (Seubert et al., Nature 361:260-263 (1993)). Other APPforms can also serve as targets for assays to assess the potential forcompounds to affect Alzheimer's disease. These include FLAPP+APPα, fulllength APP, C-terminal fragments of APP, especially C100 (the last 100amino acids of APP) and C57 to C60 (the last 57 to 60 amino acids ofAPP), and any forms of APP that include the region corresponding toAβ₁₋₄₀.

[0160] APP forms are also preferred targets for assays to assess thepotential for compounds to affect Alzheimer's disease. The absolutelevel of APP and APP transcripts, the relative levels of the differentAPP forms and their cleavage products, and localization of APPexpression or processing are all markers associated with Alzheimer'sdisease that can be used to measure the effect of treatment withpotential therapeutic compounds. The localization of APP to plaques andneuritic tissue is an especially preferred target for these assays.

[0161] Quantitative measurement can be accomplished using many standardassays. For example, transcript levels can be measured using RT-PCR andhybridization methods including RNase protection, Northern analysis, andR-dot analysis. APP and Aβ levels can be assayed by ELISA, Westernanalysis, and by comparison of immunohistochemically stained tissuesections. Immunohistochemical staining can also be used to assaylocalization of APP and Aβ to particular tissues and cell types. Suchassays were described above and specific examples are provided below.

[0162] B. Plaque-related Markers.

[0163] A variety of other molecules are also present in plaques ofindividuals with AD and in the disclosed transgenic animals, and theirpresence in plaques and neuritic tissue can be detected. The amount ofthese markers present in plaques or neuritic tissue is expected toincrease with the age of untreated transgenic animals. Preferredplaque-related markers are apolipoprotein E, glycosylation end products,amyloid P component, advanced glycosylation end products (Smith et al.,Proc. Natl. Acad. Sci. USA 91:5710 (1994)), growth inhibitory factor,laminin, collagen type IV (Kalaria and Perry (1993); Ueda et al.(1993)), receptor for advanced glycosylation products (RAGE), andubiquitin.

[0164] While the above markers can be used to detect specific componentsof plaques and neuritic tissue, the location and extent of plaques canalso be determined by using well known histochemical stains, such asCongo Red and thioflavin S, as described above and in some examplesbelow.

[0165] C. Cytoskeletal and Neuritic Markers.

[0166] Many changes in cytoskeletal markers associated with AD have alsobeen detected in transgenic PDAPP mice. These markers can be used in ADscreening assays to determine the effect of compounds on AD. Many of thechanges in cytoskeletal markers occur either in the neurofibrillarytangles or dystrophic neurites associated with plaques (Kosik et al.(1992); Lovestone and Anderton (1992); Brandan and Inestrosa (1993);Trojanowski et al. (1993); Masliah et al. (1993)).

[0167] The following are preferred cytoskeletal and neuritic markersthat exhibit changes in and/or an association with AD. These markers canbe detected, and changes can be determined, to measure the effect ofcompounds on the disclosed transgenic animals. Spectrin exhibitsincreased breakdown in AD. Tau and neurofilaments display an increase inhyperphosphorylation in AD, and levels of ubiquitin increase in AD. Tau,ubiquitin, MAP-2, neurofilaments, heparin sulfate, and chrondroitinsulphate are localized to plaques and neuritic tissue in AD and ingeneral change from the normal localization. GAP43 levels are decreasedin the hippocampus and abnormally phosphorylated tau and neurofilamentsare present in PDAPP transgenic mice.

[0168] D. Inflammatory Markers.

[0169] Alzheimer's disease is also known to stimulate animmunoinflammatory response, with a corresponding increase ininflammatory markers (Frederickson and Brunden (1994); McGeer et al.(1991); Wood et al. (1993)). The following are preferred inflammatorymarkers that exhibit changes in and/or an association with AD. Detectionof changes in these markers are useful in AD screening assays. Acutephase proteins and glial markers, such as α1-antitrypsin, C-reactiveprotein, α2-macroglobulin (Tooyama et al., Molecular & ChemicalNeuropathology 18:153-60 (1993)), glial fibrillary acidic protein(GFAP), Mac-1, F4/80, and cytokines, such as IL-1α and β, TNFα, IL-8,MIP-1α (Kim et al., J. Neuroimmunology 56:127-134 (1995)), MCP-1 (Kim etal., J. Neurological Sciences 128:28-35 (1995); Kim et al., J.Neuroimmunology 56:127-134 (1995); Wang et al., Stroke 26:661-665(1995)), and IL-6, all increase in AD and are expected to increase inthe disclosed transgenic animals. Complement markers, such as C3d, C1q,C5, C4d, C4bp, and C5a-C9, are localized in plaques and neuritic tissue.Major histocompatibility complex (MHC) glycoproteins, such as HLA-DR andHLA-A, D,C increase in AD. Microglial markers, such as CR3 receptor, MHCI, MHC II, CD 31, CD11a, CD11b, CD11c, CD68, CD45RO, CD45RD, CD18, CD59,CR4, CD45, CD64, and CD44 (Akiyama et al., Brain Research 632:249-259(1993)) increase in AD. Additional inflammatory markers useful in ADscreening assays include α2 macroglobulin receptor, Fibroblast growthfactor (Tooyama et al., Neuroscience Letters 121:155-158 (1991)), ICAM-1(Akiyama et al., Acta Neuropathologica 85:628-634 (1993)),Lactotransferrin (Kawamata et al., American Journal of Pathology142:1574-85 (1993)), C1q, C3d, C4d, C5b-9, Fc gamma RI, Fc gamma RII,CD8 (McGeer et al., Can J Neurol Sci 16:516-527 (1989)), LCA (CD45)(McGeer et al. (1989); Akiyama et al., Journal of Neuroimmunology50:195-201 (1994)), CD18 (beta-2 integrin) (Akiyama and McGeer, Journalof Neuroimmunology 30:81-93 (1990)), CD59 (McGeer et al., Brain Research544:315-319 (1991)), Vitronectic (McGeer et al., Canadian Journal ofNeurological Sciences 18:376-379 (1991); Akiyama et al., Journal ofNeuroimmunology 32:19-28 (1991)), Vitronectin receptor, Beta-3 integrin(Akiyama et al. (1991)), Apo J, clusterin (McGeer et al., Brain Research579:337-341 (1992)), type 2 plasminogen activator inhibitor (Akiyama etal., Neuroscience Letters 164:233-235 (1993)), CD44 (Akiyama et al.,Brain Research 632:249-259 (1993)), Midkine (Yasuhara et al.,Biochemical & Biophysical Research Communications 192:246-251 (1993)),Macrophage colony stimulating factor receptor (Akiyama et al., BrainResearch 639:171-174 (1994)), MRP14, 27E10, and interferon-alpha(Akiyama et al., Journal of Neuroimmunology 50:195-201 (1994)).Additional markers which are associated with inflammation or oxidativestress include 4-hydroxynonenal-protein conjugates (Uchida et al.,Biochem. Biophys. Res. Comm. 212:1068-1073 (1995); Uchida and Stadtman,Methods in Enzymology 233:371-380 (1994); Yoritaka et al., Proc. Natl.Acad. Sci. USA 93:2696-2701 (1996)), IκB, NFκB (Kaltschmidt et al.,Molecular Aspects of Medicine 14:171-190 (1993)), cPLA₂ (Stephenson etal., Neurobiology Dis. 3:51-63 (1996)), COX-2 (Chen et al., Neuroreport6:245-248 (1995)), Matrix metalloproteinases (Backstrom et al., J.Neurochemistry 58:983-992 (1992); Bignami et al., Acta Neuropathologica87:308-312 (1994); Deb and Gottschall, J. Neurochemistry 66:1641-1647(1995); Peress et al., J. Neuropathology & Experimental Neurology54:16-22 (1995)), Membrane lipid peroxidation, Protein oxidation(Hensley et al., J. Neurochemistry 65:2146-2156 (1995); Smith et al.,Proc. Natl. Acad. Sci. USA 88:10540-10543 (1991)), and diminished ATPaseactivity (Mark et al., J. Neuroscience 15:6239 (1995)). These markerscan be detected, and changes can be determined, to measure the effect ofcompounds on the disclosed transgenic animals.

[0170] E. Neuronal and Neurotransmitter-related Markers.

[0171] Changes in neuronal and neurotransmitter biochemistry have beenassociated with AD and in the disclosed PDAPP animals. In AD there is aprofound reduction in cortical and hippocampal cholinergic innervation.This is evidenced by the dramatic loss of the synthetic enzyme cholineacetyltransferase and decreased acetylcholinersterase, synaptosomalcholine uptake (as measured by hemicholinium binding) and synthesis andrelease of acetylcholine (Rylett et al. (1983); Sims et al. (1980);Coyle et al., Science 219:1184-1190 (1983); Davies and Maloney, Lancet2:1403 (1976); Perry et al., Lancet 1:189 (1977); Sims et al., J.Neurochem. 40: 503-509 (1983)) all of which are useful markers. Thesemarkers can be used in AD screening assays to determine the effect ofcompounds on AD. There is also a loss of basal forebrain neurons and thegalanin system becomes hypertrophic in AD.

[0172] In addition to changes in the markers described above in AD,there is also atrophy and loss of basal forebrain cholinergic neuronsthat project to the cortex and hippocampus (Whitehouse et al., Science215:1237-1239 (1982)), as well as alterations of entorhinal cortexneurons (Van Hoesan et al., Hippocampus 1:1-8 (1991). Based upon theseobservations measurement of these enzyme activities, neuronal size, andneuronal count numbers are expected to decrease in the disclosedtransgenic animals and are therefore useful targets for detection in ADscreening assays. Basal forebrain neurons are dependent on nerve growthfactor (NGF). Brain-derived neurotrophic factor (BDNF) may also decreasein the hippocampus in the disclosed transgenic animals and is thereforea useful target for detection in AD screening assays.

[0173] It has also been shown that APP and Aβ release are affected bystimulation of muscarinic receptors both in vitro in tissue culture aswell as in brain slices. Similar findings have also been obtained withapplication of other agonists linked to phosphoinosital turnover (Nitschet al. (1992); Hung et al., J. Biol. Chem. 268:22959-22962 (1993);Nitsch et al., Proceedings of the Eighth Meeting of the InternationalStudy Group on the Pharmacology of Memory Disorders Associated withAging 497-503 (1995); Masliah and Terry (1993); Greenamyre and Maragos(1993); McDonald and Nemeroff (1991); Mohr et al. (1994); Perry, BritishMedical Bulletin 42:63-69 (1986); Masliah et al., Brian Research574:312-316 (1992); Schwagerl et al., Journal of Neurochemistry64:443-446 (1995)). Based upon these observations, it is possible thatneurotransmitter agonists will reduce the production of Aβ in thedisclosed transgenic animals. Based on this reasoning, screening assaysthat measure the effect of compounds on neurotransmitter receptors canpossibly be used to identifying compounds useful in treating AD.

[0174] In addition to the well-documented changes in the cholinergicsystem, dysfunction in other receptor systems such as the serotinergic,adrenergic, adenosine, and nicotine receptor systems, has also beendocumented. Markers characteristic of these changes, as well as otherneuronal markers that exhibit both metabolic and structural changes inAD are listed below. Changes in the level and/or localization of thesemarkers can be measured using similar techniques as those described formeasuring and detecting the earlier markers.

[0175] The following are preferred cytoskeletal and neuritic markersthat exhibit changes in and/or an association with AD. Levels ofcathepsin (cat) D,B and Neuronal Thread Protein, and phosphorylation ofelongation factor-2, increase in AD. Cat D,B, protein kinase C, andNADPH are localized in plaque and neuritic tissue in AD. Activity and/orlevels of nicotine receptors, 5-HT₂ receptor, NMDA receptor,α2-adrenergic receptor, synaptophysin, p65, glutamine synthetase,glucose transporter, PPI kinase, drebrin, GAP43, cytochrome oxidase,heme oxygenase, calbindin, adenosine A1 receptors, mono aminemetabolites, choline acetyltransferase, acetylcholinesterase, andsymptosomal choline uptake are all reduced in AD.

[0176] Additional markers that are associated with AD or after treatmentof cells with Aβ include (1) cPLA₂ (Stephenson et al., Neurobiology ofDiseases 3:51-63 (1996)), which is upregulated in AD, (2) Hemeoxygenase-1 (Premkumar et al., J. Neurochemistry 65:1399-1402 (1995);Schipper et al., Annals of Neurology 37:758-768 (1995); Smith et al.,American Journal of Pathology 145:42-47 (1994); Smith et al., Molecular& Chemical Neuropathology 24:227-230 (1995)), c-jun (Anderson et al.,Experimental Neurology 125:286-295 (1994); Anderson et al., J.Neurochemistry 65:1487-1498 (1995)), c-fos (Anderson et al. (1994);Zhang et al., Neuroscience 46:9-21 (1992)), HSP27 (Renkawek et al., ActaNeuropathologica 87:511-519 (1994); Renkawek et al., Neuroreport 5:14-16(1993)), HSP70 (Cisse et al., Acta Neuropathologica 85:233-240 (1993)),and MAP5 (Geddes et al., J. Neuroscience Research 30:183-191 (1991);Takahashi et al., Acta Neuropathologica 81:626-631 (1991)), which areinduced in AD and in cortical cells after Aβ treatment, and (3) junb,jund, fosB, fra1 (Estus et al., J. Cell Biology 127:1717-1727 (1994)),cyclin D1 (Freeman et al., Neuron 12:343-355 (1994); Kranenburg et al.,EMBO Journal 15:46-54 (1996)), p53 (Chopp, Current Opinion in Neurology& Neurosurgery 6:6-10 (1993); Sakhi et al., Proc. Natl. Acad. Sci. USA91:7525-7529 (1994); Wood and Youle, J. Neuroscience 15:5851-5857(1995)), NGFI-A (Vaccarino et al., Molecular Brain Research 12:233-241(1992)), and NGFI-B, which are induced in cortical cells after Aβtreatment.

[0177] F. Measuring the Amounts and Localization of AD Markers.

[0178] Quantitative measurement can be accomplished using many standardassays. For example, transcript levels can be measured using RT-PCR andhybridization methods including RNase protection, Northern analysis, andR-dot analysis. Protein marker levels can be assayed by ELISA, Westernanalysis, and by comparison of immunohistochemically stained tissuesections. Immunohistochemical staining can also be used to assaylocalization of protein markers to particular tissues and cell types.The localization and the histopathological association of AD markers canbe determined by histochemical detection methods such as antibodystaining, laser scanning confocal imaging, and immunoelectronmicrography. Examples of such techniques are described in Masliah et al.(1993) and in Example 6 below.

[0179] In the case of receptors and enzymatic markers, activity of thereceptors or enzymes can be measured. For example, the activity ofneurotransmitter metabolizing enzymes such as choline acetyltransferaseand acetylcholine esterase can be measured using standard radiometricenzyme activity assays.

[0180] The activity of certain neurotransmitter receptors can bedetermined by measuring phosphoinositol (PI) turnover. This involvesmeasuring the accumulation of inositol after stimulation of the receptorwith an agonist. Useful agonists include carbachol for cholinergicreceptors and norepinephrine for glutaminergic receptors. The number ofreceptors present in brain tissue can be assessed by quantitativelymeasuring ligand binding to the receptors.

[0181] The levels and turnover of receptor ligands and neurotransmitterscan be determined by quantitative assays taken at various time points.Dopamine turnover can be measured using DOPAC and HVA. MOPEG sulfate canbe used to measure norepinephrine turnover and 5-HIAA can be used tomeasure serotonin turnover. For example, norepinephrine levels have beenshown to be reduced 20% in the hippocampus of 12 to 13 month old PDAPPtransgenic mice relative to controls. Generally, the above assays can beperformed as described in the literature, for example, in Rylett et al.(1983); Sims et al. (1980); Coyle et al., Science 219:1184-1190 (1983);Davies and Maloney, Lancet 2:1403 (1976); Perry et al., Lancet 1:189(1977); Sims et al., J. Neurochem. 40: 503-509 (1983). These markers arealso described by Bymaster et al., J. Pharm. Exp. Ther. 269:282-289(1994).

[0182] G. Screening Assays Using Cultured Cells.

[0183] Screening assays for determining the therapeutic potential ofcompounds can also be performed using cells derived from animalstransgenic for the disclosed APP constructs and cell cultures stablytransfected with the disclosed constructs. For example, such assays canbe performed on cultured cells in the following manner. Cell culturescan be transfected generally in the manner described in InternationalPatent Application No. 94/10569 and Citron et al. (1995). Derivedtransgenic cells or transfected cell cultures can then be plated inCorning 96-well plates at 1.5 to 2.5×10⁴ cells per well in Dulbecco'sminimal essential media plus 10% fetal bovine serum.

[0184] Following overnight incubation at 37° C. in an incubatorequilibrated with 10% carbon dioxide, media are removed and replacedwith media containing a compound to be tested for a two hourpretreatment period and cells were incubated as above. Stocks containingthe compound to be tested are first prepared in 100% dimethylsulfoxidesuch that at the final concentration of compound used in the treatment,the concentration of dimethylsulfoxide does not exceed 0.5%, preferablyabout 0.1%.

[0185] At the end of the pretreatment period, the media are againremoved and replaced with fresh media containing the compound to betested as above and cells are incubated for an additional 2 to 16 hours.After treatment, plates are centrifuged in a Beckman GPR at 1200 rpm forfive minutes at room temperature to pellet cellular debris from theconditioned media. From each well, 100 μL of conditioned media orappropriate dilutions thereof are transferred into an ELISA plateprecoated with antibody 266 (an antibody directed against amino acids 13to 28 of Aβ) as described in International Patent Application No.94/10569 and stored at 4° C. overnight. An ELISA assay employinglabelled antibody 6C6 (against amino acids 1 to 16 of Aβ) can be run tomeasure the amount of Aβ produced. Different capture and detectionantibodies can also be used.

[0186] Cytotoxic effects of the compounds are measured by a modificationof the method of Hansen et al., J. Immun. Method. 119:203-210 (1989). Tothe cells remaining in the tissue culture plate, 25 μL of a3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) stocksolution (5 mg/mL) is added to a final concentration of 1 mg/mL. Cellsare incubated at 37° C. for one hour, and cellular activity is stoppedby the addition of an equal volume of MTT lysis buffer (20% w/v sodiumdodecylsulfate in 50% dimethylformamide, pH 4.7). Complete extraction isachieved by overnight shaking at room temperature. The difference in theOD_(562nm) and the OD_(650nm) is measured in a Molecular Device'sUV_(max) microplate reader, or equivalent, as an indicator of thecellular viability.

[0187] The results of the Aβ ELISA are fit to a standard curve andexpressed as ng/mL Aβ. In order to normalize for cytotoxicity, theseresults are divided by the MTT results and expressed as a percentage ofthe results from a control assay run without the compound.

[0188] All publications cited herein are hereby incorporated byreference. Example 1: Expression of pMTAPP-1 in NIH3T3 and PC12 Cells.

[0189] The clone pMTAPP-1 is an example of an APP770 expressionconstruct as shown in FIG. 1a where the promoter used is themetallothionine promoter. Stable cell lines were derived by transfectingNIH3T3 and PC12 cell lines (ATCC #CCL92 and CRL1721). Five hundredthousand NIH3T3 or PC12 cells were plated into 100 mm dishes andtransfected with a mixture of 5 mg of the SalI fragment and 1 mg ofpSV2neo DNA (Southern and Berg (1982)) precipitated in the presence of50 mg lipofectin (Gibco, BRL) in a final volume of 100 μl.Polylysine-coated plates were used for PC12 cells, which normally do notadhere well to tissue culture dishes. The cells were fed with selectionmedium containing 10% fetal bovine serum in DMEM or RPMI andsupplemented with G418. Five hundred mg/ml (biological weight) and 250mg/ml of G418 were used to select colonies form NIH3T3 and PC12 cells,respectively. Fifteen days after transfection, colonies of cellsresistant to G418 were isolated by cloning rings and expanded in Tflasks. The presence of APP cDNA in the cells was detected by PCR usingthe procedure of Mullis and Faloona, Methods Enzymol. 155:335-350(1987), the teachings of which are incorporated herein.

[0190] Expression of APP in 25 colonies from each cell line was analyzedby immunostaining (Majocha et al. (1988)). Cells were grown tosubconfluence and fixed in a solution containing 4% paraformaldehyde,0.12 M NaCl, and 20 mm Na₃PO₄, pH 7.0. They were incubated overnightwith a primary monoclonal antibody against a synthetic Aβ sequence(Masters et al. (1985); Glenner and Wong) provided by Dr. RonaldMajocha, Massachusetts General Hospital, Boston, Mass., followed by ageneralized anti-mouse antibody conjugated to biotin (JacksonImmunoResearch Labs, PA). Immunostaining was then performed by addingavidin-horseradish peroxidase (HRP) (Vector Labs, Burlingame, Calif.)and diaminobenzidine as the chromogen (Majocha et al. (1985)). Theresults indicated that the pMTAPP-1 vector was expressing APP in bothNIH3T3 and PC12 cells. Example 2: Expression of pEAPP-1 in PC12 Cells.

[0191] pEAPP-1 is an example of an APP770 expression construct as shownin FIG. 1a where the promoter used is the 25 kb human APP gene promoter.DNA from this construct was transfected into PC12 cells as describedabove. Certain clones of pEAPP-1 transfected cells exhibited adifferentiation phenotype morphologically similar to that exhibited byPC12 cells treated with nerve growth factor (NGF). PC12 cells normallyare fairly round and flat cells. Those transfected with pEAPP-1 havecytoplasmic extensions resembling neurites. PC12 cells treated with NGFextend very long neuritic extensions. Thirteen PC12 cell clonestransfected with pEAPP-1 were selected and propagated. Eight of thesecell clones exhibited the spontaneous differentiation phenotype withclones 1-8, 1-1, and 1-4 exhibiting the strongest phenotypes. Stainingof pEAPP-1 transfected PC12 cells with antibody against the Aβ asdescribed in Example 1 indicated that those cells exhibiting thedifferentiation were also expressing APP. Because PC12 cells transfectedwith the pMTAPP-1 clone did not exhibit this phenotype even though theAPP770 cDNA is expressed, these results suggest that expression ofAPP770 from the human promoter has novel properties regarding thephysiology of the cell. Example 3: Expression of pMTA4 in PC12 Cells.

[0192] pMTA4 is an example of the type of construct shown in FIG. 4awhere the promoter used is the metallothionine promoter. The proteinencoded by this construct differs slightly from that depicted in FIG.4a. An APP770 cDNA clone was digested with Asp718 which cleaves afterposition 57 (number system of Kang et al. (1987)). The resulting 5′extension was filled in using the Klenow enzyme (Sambrook et al.(1989)). The same DNA preparation was also cleaved with EcoRI which alsocuts after position 2020 and the resulting 5′ extension was filled inusing the Klenow enzyme (Sambrook et al. (1989)). Self-ligation of thismolecule results in an expression clone in which the truncated proteinthus encoded contains the leader sequence, followed by a shortenedversion of the Aβ starting with the sequence Phe-Arg-Val-Gly-Ser-of theAβ followed by the 56 terminal amino acids of APP. DNA from thisconstruct was transfected into PC12 cells as described above. Example 4:Generation of Transgenic Mice expressing APP under the control of theMT-1 promoter.

[0193] Transgenic mice were made by microinjecting pMTAPP-1 vector DNAinto pronuclear embryos. pMTAPP-1 is an example of the type of constructshown in FIG. 1a in which the APP770 coding sequence is operably linkedto the metallothionine promoter. The procedures for microinjection intomouse embryos are described in Manipulating the Mouse Embryo by Hogan etal. (1986). Only a brief description of the procedures is describedbelow.

[0194] Mice were obtained from Taconic Laboratories (German Town, N.Y.).Swiss Webster female mice were used for embryo retrieval andimplantation. B6D2F₁ males were used for mating and vasectomized Swisswebster studs were used to simulate pseudopregnancy.

[0195] A. Embryo Recovery.

[0196] Female mice, 4 to 8 weeks of age, were induced to superovulatewith 5 IU of pregnant mare's serum gonadotropin (PMSG; Sigma) followed48 hours later by 5 IU of human chorionic gonadotropin (hCG; Sigma).Females were placed with males immediately after hCG injection. Embryoswere recovered from excised oviducts of mated females 21 hours after hCGin Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin(BSA; Sigma). Surrounding cumulus cells were removed with hyaluronidase(1 mg/ml). Pronuclear embryos were then washed and placed in Earle'sbalanced salt solution containing 0.4% BSA (EBSS) in a 37.5° C.incubator with a humidified atmosphere at 7% CO₂, 5% O₂, and 88% N₂until the time of injection.

[0197] B. Microinjection.

[0198] Elutip-D™ purified SalI DNA was dissolved in 5 mM Tris (pH 7.4)and 0.1 mM EDTA at 3 μg/ml concentration for microinjection.Microneedles and holding pipettes were pulled from Fisher coagulationtubes (Fisher) on a DKI model 720 pipette puller. Holding pipettes werethen broken at approximately 70 μm (O.D.) and fire polished to an I.D.of about 30 μm on a Narishige microforge (model MF-83). Pipettes weremounted on Narishige micromanipulators which were attached to a NikonDiaphot microscope. The air-filled injection pipette was filled with DNAsolution through the tip after breaking the tip against the holdingpipette. Embryos, in groups of 30 to 40, were placed in 100 μk drops ofEBBS under paraffin oil for micromanipulation. An embryo was orientedand held with the holding pipette. The injection pipette was theninserted into the male pronucleus (usually the larger one). If thepipette did not break through the membrane immediately the stage wastapped to assist in penetration. The nucleus was then injected and theinjection was monitored by swelling of the nucleus. Following injection,the group of embryos was placed in EBSS until transfer to recipientfemales.

[0199] C. Transfer.

[0200] Randomly cycling adult female mice were paired with vasectomizedSwiss Webster males. Recipient females were mated at the same time asdonor females. At the time of transfer, the females were anesthetizedwith avertin. The oviducts were exposed by a single midline dorsalincision. An incision was then made through the body wall directly overthe oviduct. The ovarian bursa was then torn with watch makers forceps.Embryos to be transferred were placed in DPBS and in the tip of atransfer pipet (about 10 to 12 embryos). The pipet tip was inserted intothe infundibulum and embryos were transferred. After the transfer, theincision was closed by two sutures.

[0201] D. Analysis of Mice for Transgene Integration.

[0202] At three weeks of age or older, tail samples about 1 cm long wereexcised for DNA analysis. The tail samples were digested by incubatingwith shaking overnight at 55° C. in the presence of 0.7 ml 5 mM Tris, pH8.0, 100 mM EDTA, 0.5% SDS and 350 μg of proteinase K. The digestedmaterial was extracted once with an equal volume of phenol and once withan equal volume of phenol:chloroform (1:1 mixture). The supernatantswere mixed with 70 μl 3 M sodium acetate (pH 6.0) and the DNA wasprecipitated by adding equal volume of 100% ethanol. The DNA was spundown in a microfuge, washed once with 70% ethanol, dried and dissolvedin 100 μl TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA).

[0203] Ten to twenty microliters of DNA from each sample was digestedwith BamHI, electrophoresed on 1% agarose gels, blotted ontonitrocellulose paper, and hybridized with ³²P-labeled APP cDNA fragment.Transgenic animals were identified by autoradiography of the hybridizednitrocellulose filters. The DNAs were also analyzed by PCR carried outby synthetic primers to generate an 800 bp fragment of APP DNA.

[0204] A total of 671 pronuclear embryos were microinjected out of which73 live and 6 dead pups were born. DNA analysis identified 9 transgenicmice (5 females and 4 males) which were bred to generate F₁ and F₂transgenics. These animals can be analyzed for expression of mRNA andprotein of APP in different tissues and for analysis of behavioral andpathological abnormalities as described above. Transgenic mice with thisconstruct express transgenic RNA. Example 5: Construction of APPconstruct containing a combination cDNA/genomic coding sequence.

[0205] A cDNA/genomic APP construct containing introns 6, 7 and 8 wasprepared by combining APP cDNA encoding exons 1-6 and 9-18 with genomicAPP sequences encoding introns 6, 7 and 8, and exons 7 and 8 (see FIG.6). In order to create a splicing cassette small enough for convenientinsertion in a pUC vector, two deletions in intronic sequences weremade. A deletion was made in intron 6 from position 143 of intron 6 tothe BamHI site located upstream of the beginning of exon 7 (1658 bpbefore the beginning of exon 7). Another deletion was made in intron 8from the first BamHI site in intron 8 to a site at 263 bp before thebeginning of exon 9. To avoid confusion, these truncated forms of APPintrons 6 and 8 are referred to herein as intron Δ6 and intron Δ8. BamHIsites were engineered at the sites of these deletions, so that they aremarked by the presence of BamHI sites. In this construct, referred to asPDAPP, exons 7 and 8 and intron 7 are intact genomic sequences, exceptthat the unique XhoI site in intron 7 was destroyed.

[0206] DNA fragments containing the truncated introns were generated asfollows: a BamHI site was engineered 143 bp into intron 6 nucleotide byPCR mutagenesis (“Mutagenesis by PCR” in PCR Technology: CurrentInnovations (Griffith and Griffith, eds., CRC Press, 1994) pages 69-83)and another BamHI site was engineered by PCR mutagenesis 263 bp prior tothe beginning of exon 9. These sites were engineered into separate APPgenomic DNA clones containing the junctions of exon 6 and intron 6, andintron 8 and exon 9, respectively, resulting in modified APP genomic DNAclones.

[0207] The entire cassette was assembled in the APP cDNA clone asfollows (FIG. 11). The 889 bp BamHI to XcmI fragment of APP cDNAcontaining exons 1 through 5 and part of exon 6 (including nucleotides 1to 843 of SEQ ID NO:5) was cloned into a vector containing BamHI andXhoI sites downstream from the insertion site to make APP770x-oligo-x.APP770x-oligo-x was then cut with XcmI and BamHI. Then two fragmentswere obtained from the modified APP genomic DNA clone containing thejunction of exon 6 and intron 6 described above by cutting with XcmI andBamHI. The resulting 34 bp fragment from the XcmI in exon 6 to the XcmIin intron 6, and 131 bp fragment from the XcmI in intron 6 to theartificially created BamHI site at position 143 bp of intron 6 wereligated into APP770x-oligo-x in a three-way ligation step to makeAPP770x-E6oligo-x. The orientation of the fragments was confirmed bysequencing. APP770x-E6oligo-x was then cut with BamHI and XhoI. Then the313 bp BamHI and XhoI fragment from the modified APP genomic DNA clonecontaining the junction of intron 8 and exon 9 was ligated intoAPP770x-E6oligo-x to make APP770xE6E9x.

[0208] APP770xE6E9x was then cut with BamHI and the 6.8 kb BamHIfragment of APP genomic DNA encoding the KPI and OX-2 domains (exons 7and 8) was inserted at this site. This fragment starts at the BamHI site1658 bp upstream of the start of exon 7 and extends to the first BamHIsite in intron 8. This BamHI fragment was obtained from a lambda phagegenomic clone encoding this portion of the APP gene, that was obtainedfrom a Human Placental genomic library in the Lambda FIXII vectorobtained from Stratagene. This BamHI fragment originally contained anXhoI site which was destroyed by cutting, filling in, and religation.The locations of the deletions are diagramed in FIG. 10. This clone,containing exons 1-8 and part of 9, and introns 6, 7 and 8, was termedthe “APP splicing cassette.” The APP splicing cassette was cut out withNruI and XhoI and used to replace the NruI to XhoI cDNA fragment of APPcDNA bearing the Hardy mutation. This mutant form of APP cDNA wasproduced by converting the G at nucleotide position 2145 to T by sitedirected mutagenesis. This changes the encoded amino acid from Val toPhe. The resulting construct is a combination cDNA/genomic APP“minigene.”

[0209] Sequencing of the 6.8 kb BamHI fragment containing APP exons 7and 8 derived from the APP genomic clone used to generate this constructshowed that intron 7 is 2.6 kb long, and that the first BamHI site inintron 8, the upstream site of the deletion in intron 8 engineered intothe APP minigene construct, is 2329 bp downstream from the end of exon8. This does not coincide with the restriction map of the APP genepublished by Yoshikai et al. (1990) and Yoshikai et al. (1991).Comparison of their map to our sequence indicates that Yoshikai et al.switched the order of two EcoRI fragments in their restriction mapping.The 1.60 kb EcoRI fragment containing exon 8 is actually upstream of the1.48 kb EcoRI fragment and the 1.48 kb EcoRI fragment Yoshikai et al.mapped in intron 7 is actually in intron 8. We have confirmed thislocation for the EcoRI fragment containing exon 8 by sizing of PCRgenerated fragments from human DNA.

[0210] This APP minigene was operatively linked to the PDGF-B promoterto provide expression of the APP cDNA/genomic construct in mammaliancells. The PDGF β-chain 5′ flanking sequence was inserted upstream ofthe NruI site at the beginning of the APP minigene. This fragmentincludes 1.3 kb upstream of the transcription initiation site, where thePDGF-B promoter resides, and approximately 70 bp of 5′ untranslatedregion, ending at the AurlI site (Higgins et al. (1994)). The late SV40polyadenylation signal, carried on a 240 bp BamHI to BclI fragment, wasadded downstream of the APP minigene. This construct, combining thePDGF-B promoter, the APP splicing cassette, the Hardy mutation, and theSV40 polyadenylation signal is referred to as PDAPP (FIG. 9).

EXAMPLE 6 Transgenic Mice Containing the PDAPP Construct.

[0211] Transgenic mice were generated using the PDAPP constructdescribed in Example 5. Transgenic mice were generated by microinjectionusing standard techniques as described above. PDAPP DNA wasmicroinjected into the embryos at the two-cell stage. Plasmid sequences(pUC) were removed by Sad and NotI digestion before microinjection.Seven founder mice were generated and line 109 was used for extensiveanalysis. Only heterozygous animals were used. Southern analysis of 104animals from four generations showed that approximately 40 copies of thetransgene were inserted at a single site and transmitted in a stablemanner. Human APP messenger RNA was produced in several tissues of thetransgenic mouse, but at especially high levels in brain. RNaseprotection assays revealed at least 20-fold more APP expression in thebrains of line 109 animals than in the mouse lines expressingneuron-specific enolase (NSE)-promoter-driven APP transgenes previouslydescribed by Quon et al. (1991), Mucke et al., Brain Res. 666:151-167(1994), McConlogue et al., Neurobiol. Aging 15:S12 (1994), and Higginset al., Ann Neurol. 35:598-607 (1994).

[0212] A. Expression of APP Transcripts and Protein.

[0213] RNA was isolated from brain tissue as described by Chomaczynskiand Sacchi, Analyt. Biochem. 162:156-159 (1987), and subjected to RT-PCRas described by Wang et al., Proc. Natl. Acad. Sci. U.S.A. 86:9717-9721(1989), using human-specific APP primers (5′-CCGATGATGACGAGGACGAT-3′,SEQ ID NO:7; 5′-TGAACACGTGACGAGGCCGA-3′, SEQ ID NO:8) using 40 cycles of1 minute at 94° C., 40 seconds at 60° C., and 50 seconds at 72° C.RT-PCR analysis demonstrated the presence of transcripts encoding the695, 751 and 770 isoforms of human APP in transgenic animal brains butnot in brains from non-transgenic littermates. The identities of thehuman APP RT-PCR bands from the transgenic mouse RNA were verified bysubcloning and sequencing.

[0214] The relative levels and alternative splicing of APP transcriptsin brains of PDAPP transgenic mice, NSE-APP transgenic mice,non-transgenic mice, and humans with and without AD were compared inRNase protection assays (Rockenstein et al., J. Biol. Chem.270:28257-28267 (1995)). PDAPP mice expressed approximately 5-foldhigher total APP mRNA levels than non-transgenic controls, and at least20-fold higher human APP mRNA levels than most NSE-APP transgenic mice.While NSE-driven human APP expression does not affect the levels ofmurine APP mRNA, PDAPP transgenic mice showed a significant 30% decreasein murine APP transcripts. While the relative abundances of murineAPP770:751:695 mRNAs in non-transgenic mouse brains were roughly 1:1:35,the corresponding human APP mRNA levels in PDAPP transgenic mouse brainswere 5:5:1.

[0215] Analysis of holo-APP was performed by brain homogenization in 10volumes of PBS containing 0.5 mM EDTA, 10 μg ml⁻¹ leupeptin and 1 mMPMSF. Samples were spun at 12,000 g for 10 min and the pelletsresuspended in RIPA (150 mM NaCl, 50 mM Tris, ph 8.0, 20 mM EDTA, 1.0%deoxycholate, 1.0% Triton X-100, 0.1% SDS, 1 mM PMSF and 10 μg ml⁻¹leupeptin). Samples (each containing 30 μg total protein) were analyzedby SDS-PAGE, transferred to Immobilon membranes and reacted with eitherthe holo-APP antibody, anti-6 (anti Bx 6), described by Oltersdorf etal., J. Biol. Chem. 285:4492-4497 (1990), or 8E5 monoclonal antibody.8E5 was prepared against a bacterial fusion protein encompassing humanAPP residues 444-692 (Oltersdorf et al. (1990)) and is human-specific,showing essentially no crossreactivity against mouse APP. Immunoblotanalysis of total APP expression (human and mouse) in transgenic mouseline 109 and control littermate brain tissue using C-terminal APPantibody anti-6 showed much higher levels of expression in thetransgenic mice. Immunoblot analysis of brain homogenates using eitherthe holo-APP polyclonal antibody anti-6 or the human-specific APPmonoclonal antibody 8E5 revealed human APP over-expression in thetransgenic mouse at levels at least 3-fold higher in hippocampus thaneither endogenous mouse APP levels or those in AD brain.

[0216] For immunoblot analysis of Aβ, a 9-month-old mouse brain washomogenized in 5 ml 6 M guanidine HCl, 50 mM Tris, pH 7.5. Thehomogenate was centrifuged at 100,000 g for 15 min and the supernatantwas dialyzed against H₂O overnight adjusted to PBS with 1 mM PMSF and 25μg ml⁻¹ leupeptin. This material was immunoprecipitated with antibody266 resin, and immunoblotted with the human-specific Aβ antibody, 6C6,as described by Seubert et al., Nature 359:325-327 (1992). Using thishuman-specific Aβ antibody (6C6), a 4 kD β amyloid-immunoreactivepeptide was isolated from the brains of the transgenic animals, whichcorresponds to the relative molecular mass of Aβ. Brain levels of Aβwere at least 10-fold higher in line 109 animals than in the previouslydescribed human APP transgenic mice. Embryonic day 16 cortical cellcultures from transgenic animals constitutively secreted human Aβ,including a substantial fraction of Aβ 1-42 (5 ng ml⁻¹ total Aβ; 0.7 ngml⁻¹ Aβ 1-42), as detected in media by human-specific Aβ enzyme-linkedimmunosorbent assays, as described by Seubert et al. (1992) andMcConlogue et al. (1994), and as described in Example 8. Thus, line-109animals greatly overexpressed human APP mRNA, holo-APP and Aβ in theirbrains.

[0217] B. Histopathology of PDAPP Transgenic Mice.

[0218] Brains from 180 transgenic and 160 age-matched non-transgenicage-matched controls (4 to 20 months old) representing five generationsof the line 109 pedigree were extensively examined histopathologically.Some mouse brains were removed and placed in alcohol fixative (Arai etal., Proc. Natl. Acad. Sci. U.S.A. 87:2249-2253 (1990)) for 48 hoursbefore paraffin embedding. Other mice were perfused with saline followedby 4% paraformaldehyde in 0.1 M sodium phosphate. For paraffin embeddedbrains, 6 μm coronal or parasaggital sections from transgenic andnon-transgenic mice were placed adjacent to each other on poly-L-lysinecoated slides. The sections were deparaffinized, rehydrated and treatedwith 0.03% H₂O₂ for 30 min before overnight incubation at 4° C. with a1:1,000 dilution of the Aβ antibody, R1280 (Tamaoka et al., Proc. Natl.Acad. Sci. U.S.A. 89:1345-1349 (1992)). For absorption studies,synthetic human Aβ 1-40 peptide (Games et al., Neurobiol. Aging13:569-576 (1992)) in 10% aqueous dimethylsulphoxide was added to afinal concentration of 7.0 μM to the diluted antibody and incubated for2 hours at 37° C. The diluent was applied to the sections and processedunder the same conditions as the standard antibody solution. Peroxidaserabbit IgG kit (Vector Labs) was then used as recommended, with3,3′-diaminobenzidine (DAB) as the chromogen. Similarly fixed human ADbrain was processed simultaneously under identical conditions.

[0219] Before 4 months of age, no obvious Aβ deposition was detected.However, by approximately 4 months of age, the transgenic animals beganto exhibit deposits of human Aβ in the hippocampus, corpus callosum andcerebral cortex. These Aβ plaques increased with age, and by eightmonths many deposits of 30 to 200 μm were seen. As the animals agedbeyond 9 months, the density of the plaques increased until theAβ-staining pattern resembled that of AD. Vascular amyloid, anotherfeature of AD pathology, developed in older mice. Robust pathology wasalso seen in another transgenic line generated from the PDAPP vector(line 35).

[0220] Aβ deposits of varying morphology were clearly evident as aresult of using a variety of Aβ antibodies, including well characterizedhuman-specific Aβ antibodies and antibodies specific for the free aminoand carboxy termini of Aβ 1-42. Antibody 9204, described by Saido etal., J. Biol. Chem. 289:15253-15257 (1994), is specific to Aβ 1-5 andwas used at a concentration of 7.0 μg ml⁻¹. Antibody 277-2, specific forAβ 1-42, was prepared by immunizing New Zealand white rabbits with thepeptide cysteine-aminoheptanoic acid-Aβ 33-42 conjugated to cationizedBSA (‘Super Carriers’; Pierce) using a standard immunization protocol(500 μg per injection). Specific antibodies were affinity-purified fromserum against the immunogen immobilized on agarose beads. Beforeincubation with antibody 277-2, sections were treated for 1 to 2 minwith 80% formic acid. For detection, the antibody was reacted using theperoxidase rabbit IgG kit (Vector Labs). The product was then visualizedusing DAB as the chromogen, Some sections were then incubated overnightat 4° C. with a 1:500 dilution of polyclonal anti-GFAP (Sigma). The GFAPantibody was reacted using the alkaline phosphatase anti-rabbit IgG kitand alkaline phosphatase substrate kit 1 (Vector Labs; used according tothe manufacturer's recommendations). Additional sections were incubatedovernight with the F480 antibody (Serotec) used at a 1:40 dilution tovisualize microglial cells. The mouse peroxidase kit (Vector Labs) wasthen used according to the manufacturer's recommendations. Some sectionswere stained with thioflavin S using standard procedures (Dickson etal., Acta Neuropath. 79:486-493 (1990)) and viewed with ultravioletlight through an FITC filter of maximum wavelength 440 nm.

[0221] Serial sections demonstrated many plaques were positively stainedwith both the 9204 and 277-2 antibodies. The forms of the Aβ depositionranged from diffuse irregular types to compacted plaques with cores.Roughly spherical, and wispy, irregular deposits, were labelled withantibody 9204 specific for the free amino terminus of Aβ. Astrocyticgliosis associated with Aβ deposition was evident after doubleimmunolabelling with antibodies to glial fibrillary acidic protein(GFAP) and human Aβ. A compacted Aβ core and ‘halo’ was evident inseveral plaques. Non-transgenic littermates showed none of theseneuropathological changes. Immunostaining was fully absorbable with therelevant synthetic peptide, and was apparent using a variety ofprocessing conditions, including fixation with paraformaldehyde andTrojanowski methods. Many plaques were stained with thioflavin S, andsome were also stained using the Bielschowsky silver method and werebirefringent with Congo Red, indicating the true amyloid nature of thesedeposits.

[0222] The majority of plaques were intimately surrounded byGFAP-positive reactive astrocytes, similar to the gliosis found in ADplaques. The neocortices of the transgenic mice contained diffuselyactivated microglial cells, as defined by their amoeboid appearance,shortened processes, and staining with Mac-1 antibody. Staining byantibodies recognizing phosphorylated neurofilaments and phosphorylatedtau indicated that aberrant phosphorylation occurred in PDAPP brain thatwas similar to AD. These phosphorylations are seen in AD and are thoughtto preclude formation of neurofibrillary tangles. Although pairedhelical filaments (PHF) have not yet been detected in PDAPP mice, thedetection of abnormally phosphorylated neurofilaments and tau arethought to be associated with, and the initial step in, the formation ofPHF in AD.

[0223] Clear evidence for neuritic pathology was apparent using bothconventional and confocal immunomicroscopy. Forty μm thick vibratomesections were incubated overnight at 4° C. with R1280 (1:1,000) incombination with polyclonal anti-synaptophysin (1:150; Dako) or 8E5 (7.0μg ⁻1). Some sections were incubated with anti-synaptophysin ormonoclonal anti-MAP 2 (1:20, Boehringer-Mannheim), and then reacted witha goat anti-rabbit biotinylated antibody (1:100) followed by a mixtureof FITC-conjugated horse anti-mouse IgG (1:75) and avidin D Texas red(1:100) (Vector Labs). The double-immunolabelled sections were viewed ona Zeiss Axiovert 35 microscope with attached laser confocal scanningsystem MRC 600 (Bio-Rad). The Texas red channel collected images of theR1280 or synaptophysin labelling, and the FITC channel collectedsynaptophysin, 8E5, or MAP 2 labelling. Optical z-sections 0.5 μm inthickness were collected from each region, similar to the imageprocessing and storage described by Masliah et al., J. Neuropath. ExpNeurol. 52:619-632 (1993).

[0224] Many Aβ plaques were closely associated with distorted neuritesthat could be detected with human APP-specific antibodies and withanti-synaptophysin antibodies, suggesting that these neurites werederived in part from axonal sprouts, as observed in the AD brain. Theplaques compressed and distorted the surrounding neuropil, also as inthe AD brain. Synaptic and dendritic density were also reduced in themolecular layer of the hippocampal dentate gyrus of the transgenic mice.This was evident by reduced immunostaining for the presynaptic markersynaptophysin and the dendritic marker MAP-2 in AD brain (Masliah etal., Am. J. Path. 138:235-246 (1991)).

[0225] Confirmation of the presence of extracellular Aβ was obtainedusing immunoelectron microscopy. For immunoelectron microscopy, micewere perfused with saline followed by 2.0% paraformaldehyde and 1.0%glutaraldehyde in cacodylate buffer. Forty μm thick vibratome sectionswere incubated with the R1280 antibody, and reacted using a peroxidaserabbit IgG kit (Vector Labs). Immunolabelled sections with Aβ depositswere then fixed in 1.0% ammonium tetraoxide and embedded inepon/araldite before viewing ultrathin sections with a Jeol CX100electron microscope (Masliah et al., Acta Neuropath. 81:428-433 (1991)).TABLE 3 Ultrastructural Similarities and Differences Between AD andPDAPP Transgenic Plaques. Alzheimer's Disease PDAPP Amyloid fibrils size9-11 mn 9-11 nm electron density moderate high pinocytic vesiclesabundant occasional Dystrophic neurites TYPE 1 dense laminar bodiesabundant abundant synaptic vesicles and contacts yes yes neurofilamentaccumulation yes yes TYPE II paired helical filaments yes none? Cellsassociated with amyloid formation microglia abundant occasional neuronsoccasional abundant neurosecretory granules abundant abundant roughendoplasmic reticulum abundant abundant coated pits yes yes

[0226] Tables 3 and 4 present a summary of the above results, showingcytological and pathological similarities between AD and PDAPP mice. Forevery feature examined, with the exception of paired helical filaments,the PDAPP mice exhibited pathology characteristic of AD. These findingsshow that production of human APP in transgenic (TG) mice is sufficientto cause not only amyloid deposition, but also many of the complexsubcellular degenerative changes associated with AD. TABLE 4 Pathologyin Alzheimer's Disease and the PDAPP Mouse. Alzheimer's Disease PDAPP AβDeposition into Plaques Diffuse + + Neuritic + + Vascular + + BrainRegion Specificity + + Neuritic Dystrophy + + Synaptic Loss + +Inflammatory Response Astrocytosis + + Microgliosis + + CytoskeletalAlterations Phosphorylated Neurofilaments + + Phosphorylated Tau + +PHF/Tangles + −(?)

[0227] The most notable feature of these transgenic mice is theirAlzheimer-like neuropathology, which includes extracellular Aβdeposition, dystrophic neuritic components, gliosis, and loss ofsynaptic density with regional specificity resembling that of AD. Plaquedensity increases with age in these transgenic mice, as it does inhumans (Selkoe, Rev. Neurosi. 17:489-517 (1994)), implying a progressiveAβ deposition that exceeds its clearance, as also proposed for AD(Maggio et al., Proc. Natl. Acad. Sci. U.S.A. 89:5462-5466 (1992)). ThePDAPP transgenic mice provide strong new evidence for the primacy of APPexpression and Aβ deposition in AD neuropathology. Such mice alsoprovide a sufficiently robust AD model in which to test whethercompounds that lower Aβ production and/or reduce its neurotoxicity invitro can produce beneficial effects in an animal model prior toadvancing such drugs into human trials. Example 7: Construction APPtransgenes expressing APP from the PDGF-B promoter.

[0228] PDAPP transgenic mice contain a splicing cassette that permitsexpression of all three major human APP isoforms, where expression isdriven by the PDGF-B promoter, and which includes a mutation in aminoacid 717, the site of familial AD mutations. It is expected that thesefeatures, and others described above, can be used independently toproduce transgenic mice useful as models of Alzheimer's disease. Somespecific examples of such constructs are described below.

[0229] A. Construction of PDAPP-wt.

[0230] A wild type version of the cDNA/genomic clone PDAPP wasconstructed in which the mutation to amino acid 717 was replaced withthe wild type. This was accomplished by replacing the 1448 bp XhoI toSpeI fragment of PDAPP, which includes the part of the APP cDNA sequencethat encodes the Hardy mutation in which Val717 is replaced by Phe, withthe 1448 bp XhoI to SpeI fragment of a wild type APP clone. Thisfragment corresponds to the region from position 1135 to 2588 of SEQ IDNO:5. None of the intron sequences of PDAPP are replaced or removed bythis substitution. This construct is referred to as PDAPP-wt. Aschematic of PDAPP-wt and its construction is shown in FIG. 12.

[0231] B. Construction of PDAPP-SwHa.

[0232] Another version of the cDNA/genomic clone PDAPP was constructedin which the Swedish mutant at amino acids 670 and 671 was introduced.Plasmid pNSE751.delta3′spl.sw contains cDNA of the human APP751 whichincludes the Swedish mutation of Lys to Asn and Met to Leu at aminoacids 670 and 671, respectively. A 563 bp EcoRI to SpeI fragment fromthis plasmid was replaced with the corresponding 563 bp EcoRI to SpeIfragment of PDAPP, which includes an identical part of the APP cDNAsequence with the exception of Phe717 of the Hardy mutation. Thisfragment corresponds to the region from position 2020 to 2588 of SEQ IDNO:5. This results in pNSE.delta3′spl.sw/ha, which contains both theSwedish mutation at amino acids 670 and 671, and the Hardy mutation atamino acid 717.

[0233] The 1448 bp XhoI to SpeI fragment of PDAPP was then replaced withthe 1448 bp XhoI to SpeI fragment of pNSE752.delta3′spl.sw/ha, whichcontains both the Swedish mutation and the Hardy mutation, to formPDAPP-Sw/Ha. A schematic of PDAPP-Sw/Ha and its construction is shown inFIG. 13.

[0234] C. Construction of PDAPP695_(V-F).

[0235] A construct encoding only APP695, but retaining the Hardymutation, PDGF-B promoter, and vector sequences of PDAPP, can be made.This can be accomplished by ligating the 6.6 kb XhoI to NruI fragmentfrom PDAPP, which contains the C-terminal part of the APP sequences, andthe polyadenylation, pUC, and PDGF-B promoter sequences, to the 1.2 kbXhoI to BclI fragment of pCK695, which contains a hybrid splice signaland the remaining N-terminal portion of the APP sequences (on a 911 bpXhoI to NruI fragment of APP695 cDNA). The hybrid splice signal is thesame as was described earlier and is also present in vector pohCK751,which is described by Dugan et al., J Biological Chem. 270:10982-10989(1995). pCK695 is identical to pohCK751 except that the herpes simplesvirus replication and packaging sequences of pohCK751 were removed, andthe plasmid encodes APP695 instead of APP751.

[0236] In this vector the PDGF-B promoter drives the expression ofAPP695 containing the mutation of Val717 to Phe. The hybrid splicesignal is included to potentially enhance expression. Additional vectorsderived from this may be constructed which lack any splice signals, orinto which other splice signals have been added to obtain this samefunction.

[0237] D. Construction of PDAPP751_(V-F).

[0238] A construct encoding only APP751, but retaining the Hardymutation, PDGF-B promoter, and vector sequences of PDAPP, can be made.This can be accomplished by ligating the 6.65 kb XhoI to KpnI fragmentof PDAPP, including part of the APP sequences, the polyadenylationsignals, pUC and PDGF-B promoter sequences to the 1.0 kb KpnI to XhoIfragment containing the remainder of the human APP751 cDNA sequences(nucleotides 57 to 1084 of SEQ ID NO:3) to make the intermediate plasmidPDAPPδsp751_(V-F). The 1.0 kb KpnI to XhoI fragment encoding a portionof human APP751 can be obtained from the plasmid poCK751, which isidentical to pohCK751 except that the herpes simplex viral sequenceswere removed.

[0239] To introduce splicing sequences, the first intron from PDAPP,which is intron Δ6, is then inserted into PDAPPδsp751_(V-F) to makePDAPP751_(V-F). To accomplish this, the 2,758 bp Asp718 to ScaI fragmentof PDAPP containing exons 2 through 6, intron Δ6, and part of exon 7, isligated to the 6,736 bp fragment obtained by complete digestion ofPDAPPδsp751_(V-F) with Asp718 and partial digestion with ScaI. This6,736 bp fragment provides the remaining additional APP sequences (partof exon 1, the rest of exon 7, and exons 9 through 18), polyadenylationsignals, pUC and PDGF-B promoter sequences. The resulting construct isreferred to as PDAPP751_(V-F).

[0240] In this vector the PDGF-B promoter drives the expression ofAPP751 containing the mutation of Val717 to Phe. One splice signal(derived from intron 6) is included to potentially enhance expression.Additional vectors derived from this may be constructed which lack anysplice signals, or into which other splice signals have been added toobtain this same function.

[0241] E. Construction of PDAPP770_(V-F).

[0242] A construct encoding only APP770, but retaining the Hardymutation, PDGF-B promoter, and vector sequences of PDAPP, can be made.This can be accomplished by replacing the KpnI to XhoI fragment ofPDAPP751_(V-F) containing APP exons 2-7 and a part of exon 9, with theKpnI to XhoI fragment of APP770 cDNA, which contains exons 2-8 and apart of exon 9. This fragment corresponds to nucleotides 57 to 1140 ofSEQ ID NO:5. The resulting construct is referred to as PDAPP770_(V-F).

[0243] In this vector the PDGF-B promoter drives the expression ofAPP770 containing the mutation of Val717 to Phe. PDAPP770_(V-F) containsthe same intron sequences present in PDAPP751_(V-F). Additional vectorsderived from this may be constructed into which a splice signals havebeen added to obtain enhanced expression. Example 8: Expression Levelsof APP Expression Products in Brain Tissue of PDAPP Mice.

[0244] The PDAPP mouse line described in Example 6 was examined for thelevels of several derivatives of the APP in hippocampal, cortical, andcerebellar brain regions of mice of various ages. Levels of APP cleavedat the beta-secretase site (APPβ) and APP containing at least 12 aminoacids of Aβ (FLAPP+APPα; a mixture of APPα and full length APP (FLAPP))were found to be nearly constant within a given brain region at all agesevaluated. The hippocampus expressed the highest level of all APP forms.In contrast, guanidine extractable levels of Aβ showed remarkableage-dependent increases in a manner that mirrored the amyloid plaquedeposition observed immunohistochemically. Specifically, Aβ levels inhippocampus increased 17-fold by 8 months of age and 106-fold by 1 yearof age, compared to that found in 4 month old animals. At 1 year of ageAβ constitutes approximately 1% of the total protein in hippocampus. Thecerebral cortex also showed large increases in Aβ with age. In contrast,the mean level of Aβ in cerebellum across all age groups wascomparatively low and unchanging.

[0245] Further analysis of the Aβ in these brains using an ELISAspecific for Aβ₁₋₄₂ showed that this longer version made up 27% of the19 pmoles/g of the Aβ present in the brains of young animals; thispercentage increased to 97% of the 690 pmoles/g in 12 month old animals.The selective deposition of Aβ₁₋₄₂ and the spacial distribution of theAβ deposits are further evidence that the pathological processes ongoingin the PDAPP transgenic mice parallel the human Alzheimer's diseasedcondition.

[0246] Levels of Aβ-containing proteins were measured through the use ofELISAs configured with antibodies specific to Aβ, Aβ₁₋₄₂, APP cleaved atthe β-secretase site (Seubert et al. (1993)), and APP containing thefirst 12 amino acids of Aβ (FLAPP+APPα; a mixture of full length APP andα-secretase cleaved APP (Esch et al.)). Striking similarities in boththe regional variation and depositing form of Aβ are noted between themouse model and the human AD condition. The results also show that,because of the magnitude and temporal predictability of Aβ deposition,the PDAPP mouse is a practical model in which to test agents that eitherinhibit the processing of APP to Aβ or retard Aβ amyloidosis.

[0247] A. Materials and Methods.

[0248] 1. Brain Tissue Preparation.

[0249] The heterozygote transgenic (Line 109, Games et al.; Rockensteinet al.) and non-transgenic animals were anesthetized with Nembutol (1:5solution in 0.9% saline) and perfused intracardially with ice cold 0.9%saline. The brain was removed and one hemisphere was prepared forimmunohistochemical analysis, while four brain regions (cerebellum,hippocampus, thalamus, and cortex) were dissected from the otherhemisphere and used for Aβ and APP measures.

[0250] To prepare tissue for ELISAs, each brain region was homogenizedin 10 volumes of ice cold guanidine buffer (5.0 M guanidine-HCl, 50 mMTris-Cl, pH 8.0) using a motorized pestle (Kontes). The homogenates weregently mixed on a Nutator for three to four hours at room temperature,then either assayed directly or stored at −20° C. prior to quantitationof Aβ and APP. Preliminary experiments showed the analytes were stableto this storage condition.

[0251] 2. Aβ Measurements.

[0252] The brain homogenates were further diluted 1:10 with ice-coldcasein buffer (0.25% casein, phosphate buffered saline (PBS), 0.05%sodium azide, 20 μg/ml aprotinin, 5 mM EDTA pH 8.0, 10 μg/ml leupeptin),reducing the final concentration of guanidine to 0.5 M, beforecentrifugation (16,000× g for 20 minutes at 4° C.). The Aβ standards(1-40 or 1-42 amino acids) were prepared such that the final compositionincluded 0.5 M guanidine in the presence of 0.1% bovine serum albumin(BSA).

[0253] The “total” Aβ sandwich ELISA consists of two monoclonalantibodies (mAb) to Aβ. The capture antibody, 266, is specific to aminoacids 13-28 of Aβ (Seubert et al. (1992)); while the antibody 3D6, whichis specific to amino acids 1-5 of Aβ, was biotinylated and served as thereporter antibody. The 3D6 biotinylation procedure employs themanufacturer's (Pierce) protocol for NHS-biotin labeling ofimmunoglobulins except 100 mM sodium bicarbonate, pH 8.5 buffer wasused. The 3D6 antibody does not recognize secreted APP or full-lengthAPP but detects only Aβ species with amino terminal aspartic acid. Theassay has a lower limit of sensitivity of approximately 50 pg/ml (11.4pM) and showed no cross-reactivity to the endogenous murine Aβ peptideat concentrations up to 1 ng/ml.

[0254] The configuration of the Aβ₁₋₄₂-specific sandwich ELISA employsthe mAb 21F12, which was generated against amino acids 33-42 of Aβ. Theantibody shows less than 0.4% cross-reactivity with Aβ₁₋₄₀ in eitherELISA or competitive radioimmunoassay (RIA). Biotinylated 3D6 is alsothe reporter antibody in this assay which has a lower limit ofsensitivity of approximately 125 pg/ml (28.4 pM).

[0255] The 266 and 21F12 mAbs were coated at 10 μg/ml into 96-wellimmunoassay plates (Costar) overnight at room temperature. The plateswere then aspirated and blocked with 0.25% human serum albumin in PBSbuffer for at least 1 hour at room temperature, then stored desiccatedat 4° C. until use. The plates were rehydrated with wash buffer prior touse. The samples and standards were added to the plates and incubated atroom temperature for 1 hour. The plates were washed at least 3 timeswith wash buffer (Tris buffered saline, 0.05% Tween 20) between eachstep of the assay.

[0256] The biotinylated 3D6, diluted to 0.5 μg/ml in casein assay buffer(0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was incubated in the wellfor 1 hour at room temperature. Avidin-HRP (Vector, Burlingame, Calif.),diluted 1:4000 in casein assay buffer, was added to the wells andincubated for 1 hour at room temperature. The calorimetric substrate(100 μl), Slow TMB-ELISA (Pierce), was added and allowed to react for 15minutes, after which the enzymatic reaction is stopped with 25 μl of 2 NH₂SO₄. Reaction product was quantified using a Molecular Devices Vmaxmeasuring the difference in absorbance at 450 nm and 650 nm.

[0257] 3. APP ELISAs.

[0258] Two different APP assays were utilized. The first recognizes APPαand full length forms of APP (FLAPP+APPα), while the second recognizesAPPβ (APP ending at the methionine preceding the Aβ domain (Seubert etal. (1993)). The capture antibody for both the FLAPP+APPα and APPβassays is 8E5, a monoclonal antibody raised to a bacterially expressedfusion protein corresponding to human APP amino acids 444-592 (Games etal.). The reporter mAb (2H3) for the FLAPP+APPα assay was generatedagainst amino acids 1-12 of Aβ. The lower limit of sensitivity for the8E5/2H3 assay is approximately 11 ng/ml (150 pM). For the APPβ assay,the polyclonal antibody 192 was used as the reporter. This antibody hasthe same specificity as antibody 92 (Seubert et al. (1993)), that is, itis specific to the carboxy-terminus of the β-secretase cleavage site ofAPP. The lower limit of sensitivity for the β-secretase 8E5/192 assay isapproximately 43 ng/ml (600 pM).

[0259] For both APP assays, the 8E5 mAb was coated onto 96-well Costarplates as described above for 266. Purified recombinant secreted APPα(the APP751 form) and APP596 were the reference standards used for theFLAPP+APPα and APPβ assays, respectively. APP was purified as describedpreviously (Esch et al.) and APP concentrations were determined by aminoacid analysis. The 5 M guanidine brain homogenate samples were diluted1:10 in specimen diluent for a final buffer composition of 0.5 M NaCl,0.1% NP-40, 0.5 M guanidine. The APP standards for the respective assayswere diluted into buffer of the same final composition as for thesamples. The APP standards and samples were added to the plate andincubated for 1.5 hours at room temperature. The plates were thoroughlywashed between each step of the assay with wash buffer. Reporterantibodies 2H3 and 192 were biotinylated following the same procedure asfor 3D6 and were incubated with samples for 1 hour at room temperature.Streptavidin-alkaline phosphatase (Boehringer Mannheim), diluted 1:1000in specimen diluent, was incubated in the wells for 1 hour at roomtemperature. The fluorescent substrate 4-methyl-umbellipheryl-phosphate,was added, and the plates read on a Cytofluor™ 2350 (Millipore) at 365nm excitation and 450 nm emission.

[0260] 4. Monoclonal Antibody Production.

[0261] The immunogens for 3D6, 21F12, and 2H3 were separately conjugatedto sheep anti-mouse immunoglobulin (Jackson Immunoresearch Labs) usingmaleimidohexanoyl-N-hydroxysuccinimide (Pierce). A/J mice (JacksonLaboratories) were given intraperitoneal injections (IP) of 100 μg ofthe appropriate immunogen emulsified in Freund's complete adjuvant(Sigma) and two subsequent IP injections of 100 μg immunogen were givenon a biweekly basis in Freund's incomplete adjuvant (Sigma). Two tothree weeks after the third boost, the highest titer mouse of a givenimmunogen was injected intravenously and intraperitoneally with 50-100μg of immunogen in PBS. Three days post injection, the spleen wasremoved, splenocytes were isolated and fused with SP2/0-Ag14 mousemyeloma cells. The hybridoma supernatants were screened for highaffinity monoclonal antibodies by RIA as previously described (Seubertet al. (1992)). Purified monoclonal antibodies were prepared fromascites.

[0262] 5. Immunohistochemistry.

[0263] The tissue from one brain hemisphere of each mouse was drop-fixedin 4% paraformaldehyde and post-fixed for three days. The tissue wasmounted coronally and 40 μm sections were collected using a vibratome.The sections were stored in anti-freeze at −20° C. prior to staining.Every sixth section, from the posterior part of the cortex through thehippocampus, was immunostained with biotinylated 3D6 at 4° C.,overnight. The sections were then incubated with horseradish peroxidaseavidin-biotin complex (Vector) and developed using 3,3′-diaminobenzidine(DAB) as the chromogen.

[0264] B. Results.

[0265] 1. Aβ and APP Assays.

[0266] The FLAPP+APPα assay recognizes secreted APP including the first12 amino acids of Aβ. Since the reporter antibody (2H3₁₋₁₂) is notspecific to the alpha clip site occurring between Aβ amino acids 16 and17 (Esch et al.), this assay also recognizes full length APP.Preliminary experiments using immobilized APP antibodies to thecytoplasmic tail of full length APP to deplete the mixture suggest thatapproximately 30 to 40% of the FLAPP+APPα is full length. The APPβ assayrecognizes only the APP clipped immediately amino-terminal to the Aβregion due to the specificity of the polyclonal reporter antibody 192(Seubert et al. (1993)).

[0267] The specific nature of the Aβ immunoreactivity was furthercharacterized as follows. Guanidine homogenates of brain (excludingcerebellum and brain stem) were subjected to size exclusionchromatography (Superose 12) and the resulting fractions analyzed usingthe total Aβ assay. Comparisons were made of 2, 4, and 12 monthtransgenic mouse brain homogenates and a non-transgenic mouse brainhomogenate to which Aβ₁₋₄₀, had been spiked at a level roughly equal tothat found in the 12 month old transgenic mice. The elution profiles ofthe transgenic brain homogenate were similar in that the peak fractionsof Aβ immunoreactivity occurred in the same position, a single broadsymmetric peak which was coincident with the immunoreactive peak ofspiked Aβ₁₋₄₀. Attempts were then made to immunodeplete the Aβimmunoreactivity using resin bound antibodies against Aβ (mAb 266against Aβ₁₃₋₂₈), the secreted forms of APP (mAb 8E5 against APP₄₄₄₋₅₉₃of the APP695 form), the carboxy-terminus of APP (mAb 13G8 againstAPP₆₇₆₋₆₉₅ of the APP695 form), or heparin agarose. Only the 266 resincaptured Aβ immunoreactivity, demonstrating that full length APP orcarboxy-terminal fragments of APP are not contributing to the Aβmeasurement. The Aβ₁₋₄₂ ELISA employs a capture antibody that recognizesAβ₁₋₄₂ but not Aβ₁₋₄₀. The Aβ₁₋₄₂ assay, like the total Aβ assay, is notaffected by the full length or carboxy-terminal forms of APP containingthe Aβ region in the homogenates as shown by similar immunodepletionstudies.

[0268] 2. Total Aβ and APP Measures.

[0269] Table 5 shows the levels of total Aβ, FLAPP+APPα, and APPβ in thehippocampus, cortex, cerebellum, and thalamus of transgenic mice as afunction of age. Each data point represents the mean value for each agegroup. The relative levels of FLAPP+APPα and APPβ in all four brainregions remain relatively constant over time. The hippocampus expressesthe highest levels of FLAPP+APPα and APPβ followed by the thalamus,cortex, and cerebellum, respectively. In the hippocampus, the levels ofFLAPP+APPα are approximately 3.5 to 4.5-fold higher than APPβ at allages. The mean value of all ages for FLAPP+APPα and APPβ assays in thehippocampus were 674 (±465) pmoles/gram and 175 (±11) pmoles/gram,respectively. From this it can be estimated that the pool of brain APPconsists of approximately 50% APPα, 30% full length APP, and 20% APPβ.TABLE 5 PDAPP Transgene Cohort Animal Data Total Aβ & APP Measures inpmoles/gram of Brain Tissue. AGE IN Aβ & APP MONTHS FORM CEREBELLUMHIPPOCAMPUS CORTEX THALAMUS 2 Aβ 4.03 ± 1.08 35.41 ± 6.38 14.25 ± 2.276.41 ± 1.59 (n = 8) (n = 8) (n = 8) (n = 8) 2 FLAPP + ND ND ND ND APPα 2APPβ ND ND ND ND 4 Aβ 4.10 ± 0.61 38.08 ± 6.51 15.95 ± 2.60 7.60 ± 1.52(n = 14) (n = 14) (n = 14) (n = 14) 4 FLAPP + 395 ± 120 703 ± 106 446 ±70 6.37 ± 166 APPα (n = 14) (n = 14) (n = 14) (n = 14) 4 APPβ 78 ± 38198 ± 30 126 ± 23 70 ± 17 (n = 14) (n-14) (n = 14) (n = 14) 6 Aβ 4.55 ±1.38 87.48 ± 30.33 30.19 ± 8.33 8.34 ± 2.40 (n = 10) (n = 10) (n = 10)(n = 10) 6 FLAPP + 403 ± 77 694 ± 107 506 ± 97 670 ± 156 APPα (n = 10)(n = 10) (n = 10) (n = 10) 6 APPβ 51 ± 87 194 ± 35 129 ± 25 56 ± 33 (n =10) (n = 10) (n = 10) (n = 10) 6.5 Aβ 5.42 ± 1.08 133.63 ± 57.10 33.27 ±12.19 8.83 ± 1.19 (n = 10) (n = 10) (n = 10) (n = 10) 6.5 FLAPP + 346 ±74 580 ± 115 436 ± 63 553 ± 123 APPα (n = 10) (n = 10) (n = 10) (n = 10)6.5 APPβ 27 ± 77 169 ± 41 108 ± 16 58 ± 22 (n = 10) (n = 10) (n = 10) (n= 10) 7 Aβ 4.44 ± 0.56 200.77 ± 94.68 60.55 ± 27.13 8.94 ± 1.19 (n = 10)(n = 10) (n = 10) (n = 10) 7 FLAPP + 378 ± 70 656 ± 73 469 ± 62 604 ±107 APPα (n = 10) (n = 10) (n = 10) (n = 10) 7 APPβ 56 ± 52 176 ± 27 101± 20 56 ± 28 (n = 10) (n = 10) (n = 10) (n = 10) 7.5 Aβ 5.14 ± 1.39461.35 ± 345.95 81.839 ± 53.00 10.84 ± 5.22 (n = 10) (n = 10) (n = 10)(n = 10) 7.5 FLAPP + 362 ± 54 554 ± 77 409 ± 44 503 ± 80 APPα (n = 10)(n = 10) (n = 10) (n = 10) 7.5 APPβ 20 ± 58 168 ± 27 118 ± 21 57 ± 22 (n= 10) (n = 10) (n = 10) (n = 10) 8 Aβ 4.42 ± 0.73 635.52 ± 302.45 128.68± 62.80 10.87 ± 3.39 (n = 13) (n = 13) (n = 13) (n = 13) 8 FLAPP + 386 ±52 660 ± 102 494 ± 87 672 ± 150 APPα (n = 13) (n = 13) (n = 13) (n = 13)8 APPβ 64 ± 77 174 ± 27 102 ± 26 57 ± 30 (n = 13) (n = 13) (n = 13) (n =13) 8.5 Aβ 5.54 ± 1.11 633.11 ± 363.14 118.39 ± 59.91 13.96 ± 7.34 (n =10) (n = 10) (n = 10) (n = 10) 8.5 FLAPP + 439 ± 79 764 ± 114 558 ± 80750 ± 132 APPα (n = 10) (n = 10) (n = 10) (n = 10) 8.5 APPβ 28 ± 59 185± 34 108 ± 42 47 ± 28 (n = 10) (n = 10) (n = 10) (n = 10) 9 Aβ 5.52 ±1.11 1512.39 ± 624.286 254.83 ± 105.927 19.46 ± 8.99 (n = 10) (n = 10)(n = 10) (n = 10) 9 FLAPP + 500 ± 112 763 ± 125 549 ± 78 815 ± 167 APPα(n = 10) (n = 10) (n = 10) (n = 10) 9 APPβ 4 ± 83 169 ± 25 121 ± 32 49 ±26 (n = 10) (n = 10) (n = 10) (n = 10) 10 Aβ 4.04 ± 1.02 2182.21 ±1194.49 343.49 ± 165.531 15.46 ± 13.38 (n = 11) (n = 11) (n = 11) (n =11) 10 FLAPP + 452 ± 130 678 ± 93 491 ± 102 693 ± 166 APPα (n = 11) (n =11) (n = 11) (n = 11) 10 APPβ 52 ± 32 159 ± 22 87 ± 15 46 ± 10 (n = 11)(n = 11) (n = 11) (n = 11) 12 Aβ 3.26 ± 0.35 4356.23 ± 1666.44 691.17 ±360.93 18.08 ± 13.50 (n = 9) (n = 9) (n = 9) (n = 9) 12 FLAPP + 385 ±166 638 ± 272 444 ± 171 708 ± 278 APPα (n = 10) (n = 10) (n = 10) (n =10) 12 APPβ 41 ± 29 134 ± 47 76 ± 31 35 ± 19 (n = 10) (n = 10) (n = 10)(n = 10)

[0270] In contrast to APP levels, Aβ levels increased dramatically withage in the hippocampus and cortex. However, no such increase was notedin the cerebellum of the PDAPP transgenic mice, and only a moderateincrease was seen in thalamus (Table 5). The increase of Aβ is greaterin the hippocampus relative to the cortex, which also correlates withthe 3D6 immunohistochemical results (see discussion below). Compared tothe cortex levels of 4 month old mice, Aβ levels increase 10-fold by 8months of age and 41-fold at 12 months old (660±380 pmoles Aβ/gramtissue at age 12 months). The corresponding increases in Aβ observed inhippocampus are even more impressive, as the 8 month value is 15 timesthat at 4 months old and increases to 106-fold at 12 months old(4,040±1750 pmoles Aβ/g tissue at 12 months). At 12 months of age, Aβconstitutes approximately 1% of protein in hippocampus of the PDAPPmice.

[0271] To see if the dramatic rise in brain Aβ concentration is due toamyloid deposition, we next visualized Aβ depositsimmunohistochemically, using the opposite hemisphere of the same miceused for Aβ measurements. Notably, a parallel increase in Aβ plaqueburden and Aβ level exists. These findings strongly argue that the risein brain Aβ concentration determined by ELISA is due to theage-dependent amyloidosis.

[0272] 3. Aβ₁₋₄₂ Measures in Transgenic Mouse Brain.

[0273] Concentrations of Aβ₁₋₄₂ in the cortex of transgenic mice wereevaluated at different ages. As shown in Table 6, the percentage of Aβwhich is Aβ₁₋₄₂ in the cortex of transgenic mice, also increases withage. The ELISA data suggest that Aβ₁₋₄₂ is preferentially depositing inthe transgenic mice, and that the deposits detected by mAb 3D6immunostaining are primarily Aβ₁₋₄₂. TABLE 6 Aβ¹⁻⁴² Levels in the Cortexof Transgenic Brain. Age (months) Aβ¹⁻⁴² (pmoles/g) 4 4.71 8 75.65 10247.43 12 614.53

[0274] 4. Aβ Immunostaining in PDAPP Transgenic Brain.

[0275] Transgenic animals with Aβ values representing the mean Aβ valueof the age group were used for 3D6 immunostaining. A progression of Aβdeposition is seen in the 4, 8, 10, and 12 months old animals. At fourmonths of age, transgenic brains contained small, rare punctatedeposits, 20 μm in diameter, that were only infrequently observed in thehippocampus and frontal and cingulate cortex. By eight months of age,these regions contained a number of thioflavin-positive Aβ aggregatesthat formed plaques as large as 150 μm in diameter. At ten months ofage, many large Aβ deposits were found throughout the frontal andcingulate cortex, and the molecular layers of the hippocampus. The outermolecular layer of the dentate gyrus receiving perforant pathwayafferents from the entorhinal cortex was clearly delineated by Aβdeposition. This general pattern was more pronounced by heavier Aβdeposition at one year of age. The anatomical localization of Aβdeposition is remarkably similar to that seen in Alzheimer's disease.

[0276] C. Discussion.

[0277] Aβ amyloidosis is an established diagnostic criteria ofAlzheimer's disease (Mirra et al., Neurology 41:479-486 (1991)) and isconsistently seen in higher cortical areas as well as the hippocampalformation of the brain in affected subjects. It is believed that Aβamyloidosis is a relatively early event in the pathogenesis of AD thatsubsequently leads to neuronal dysfunction and dementia through acomplex cascade of events (Mann et al., Neurodegeneration 1:201-215(1992); Morris et al., Neurology 46:707-719 (1996)). Various pathways ofAPP processing have been described (reviewed in Schenk et al., J. Med.Chem. 38:4141-4154 (1995)) including the major a-secretase pathway wherecleavage of APP occurs with Aβ (Esch et al.) and the amyloidogenic orβ-secretase pathway where cleavage of APP occurs at the N-terminus of Aβ(Seubert et al. (1993)). Further cleavage of APP leads to theconstitutive production of Aβ forms including those ending at position40 (Aβ₁₋₄₀) or 42 (Aβ₁₋₄₂). ELISAs that detect specific APP productsarising from these individual pathways in the PDAPP mouse brain allowdetermination of whether differential processing of APP contributes tothe regional or temporal specificity of amyloid formation anddeposition.

[0278] Aβ amyloid deposition seen in the PDAPP mouse brain is highly ageand region specific. Amyloid deposition begins at around 7 months ofage, and by 12 months of age, amyloid deposition is very profoundthroughout the hippocampus and in the rostral region of the cortex. Theage dependent increases in amyloid deposition correlate well with thedramatic rise in Aβ levels in these brain regions as measured by ELISAassay. An increase in Aβ is measurable by 7 months of age and by 10months the hippocampus as 2180 pmoles/g of Aβ, a concentrationequivalent to that of my cytoskeletal proteins and comparable to thelevels found in the cortex of human AD brain (Gravina et al., J. Biol.Chem. 270:7013-7016 (1995)). Aβ levels in the cerebellum, an unaffectedbrain region, still are at 4 pmoles/g—essentially unchanged relative tothe levels at 4 months of age—again correlating with amyloid depositionmeasured by histological analyses. These results indicate that in agedPDAPP mice, brain Aβ levels reflects amyloid burden and therefore directimmunoassay measurement of brain Aβ levels can be used to test forcompounds that reduce amyloid plaque burden.

[0279] In the PDAPP mouse, individuals suffering Down's Syndrome, andindividuals with certain forms of FAD, overproduction of Aβ is almostcertainly an accelerating factor not only in Aβ deposition but insubsequent neuropathology (Citron et al., Mann et al., Miller et al.,Archives of Biochem. Biophys. 301:41-52 (1993)). A comparison of the Aβmeasurements seen in the PDAPP mouse with those reported for AD braintissue reveals several striking similarities. For example, in the PDAPPmouse, the relative levels of Aβ peptide in hippocampus from young (2months of age) versus old (10 months of age) mice is nearly a hundredfold. Similar findings were noted by Gravina et al. in comparing controlbrain tissue relative to that of AD. The rise in brain Aβ levels in thePDAPP mouse is rather pronounced between the ages of six to nine monthsof age. Again, this timecourse parallels, in an accelerated manner, thatseen in Down's Syndrome brain tissue, where amyloid deposition begins atapproximately 30 years of age and increases substantially untilapproximately age 60 (Mann).

[0280] In summary, the above results show that a reproducible increasein measurable Aβ occurs in the brain tissue of the PDAPP mice and thatthis increase correlates with the severity of amyloid deposition. Thesefindings indicate that these mice can be used to identify agents orcompounds that pharmacologically reduce Aβ peptide production or affectits deposition. Example 9: Behavioral Differences in PDAPP TransgenicMice.

[0281] Alzheimer's disease is characterized by cognitive deficitsincluding memory loss, and impairment of memory functions. To determineif the disclosed transgenic mice exhibit similar deficits, transgenic(TG) and non-transgenic (nTG) mice were evaluated for task performancein three types of maze apparatus used to test working and referencememory; the Y maze, the radial arm maze (RAM), and the water maze. Thetransgenic mice tested represent the fifth generation derived from thePDAPP mice described in Example 6. The Y maze and the radial arm mazeare used to assess spontaneous alternation which is a function ofworking memory. For the Y maze task, the mouse is placed in the stem ofa Y maze twice, each time allowing a choice entry into one of the arms.Entering both arms is a successful alternation, requiring memory of thepreviously entered arm, while entering the same arm on both trials is afailure. Chance performance is 50% alternation, that is, 50% of the micealternate.

[0282] For the radial arm maze task, the mouse is placed at the centerof a maze with multiple arms radiating from the center. In the testingdescribed below, a radial eight-arm maze was used. Alternationperformance is measured by allowing only eight entries, with the numberof different arms entered being the measure of performance. The numberof different-arm entries can be compared to the number of different-armentries expected by chance, which is 5.25 (Spetch and Wilkie, “A programthat stimulates random choices in radial arm mazes and similar choicesituations” Behavior Research Methods & Instrumentation 12:377-378(1980)). Performance above chance, that is, above 5.25, requires memoryof the previously entered arms.

[0283] The water maze used for the tests described below consists of apool of water in which a submerged platform is placed. This hiddenplatform (HP) can be found by swimming mice either by chance (firsttrial) or through memory of positional clues visible from the tank(subsequent trials). Subject mice were trained in the hidden platformtask according to standard procedures. Briefly, mice were firstpretrained in a small pool (47 cm diameter, 20 cm platform), whichteaches them how to navigate in water, that the platform is the goal,that there is no other escape, and that to find it they must resisttheir natural inclination to stay along the sides of the pool. They werethen trained to find a single platform position in the hidden platformtask using a larger pool and smaller platform (71 cm pool, 9 cmplatform).

[0284] During the HP task, visual cues were located inside the pool(intramaze cues; black pieces of cardboard—circle, plus, or horizontallines—located in three quadrants at the top of the wall, which was 38 cmhigh above water level), and various room cues were visible outside thepool (extramaze cues).

[0285] The mice assigned to the characterization cohort study weretested on the behavioral tasks described above over 3 days during theweek or two before euthanasia. Their transgenic status was not known tothe tester. Non-transgenic littermates were used for comparison. Eachmorning the subject mice were run in the Y maze and RAM as describedabove. They were then tested for general strength on the inclined plane(INP) test. For this, mice were placed in a 10-cm-wide runway lined withridged plastic and elevated with the head up at 35°. The angle was thenincreased until the mouse slid off, and the angle was recorded. This wasrepeated three times each day. The average scores for the three dayswere calculated for each mouse for the Y maze (0=repeat, 1=alternate),RAM (number of different arms and time to finish, 10 minute limit), andINP (average of all nine trials). General activity was also rated on thefirst day of testing. Each mouse was observed in the cage, and picked upand held. A mouse that remained calmly in the hand was scored 1, withprogressively greater activity and reaction to handling scored up to 4.

[0286] Following the above tests each day, mice were tested in the watermaze as described above. Briefly, mice were pretrained in a small poolto climb on a large submerged platform as their only means of escapefrom the water. They were then given six blocks of four trials each tolearn the location of a small platform in a large pool. For analysis,all four trials within each block were averaged. The exception was thefirst hidden platform block, for which only the last three trials wereaveraged. The first trial was analyzed separately, because it is theonly one for which platform location could not be known, and thus didnot relate to spatial learning. It is thus used as a control fornon-spatial factors, such as motivation and swimming speed. Theperformance effects between blocks were analyzed as a repeated measurefor the hidden platform task. Standard analysis of variance (ANOVA)calculations were used to assess the significance of the results.

[0287] Results in the RAM show that TG performed significantly worsethan nTG across all ages (Group effect: p=0.00006). The time to finishwas also significantly different between TG and nTG mice (Group effect:p=0.005). The correlation between the time to finish and the number ofarms chosen was small (R=−0.15, p=0.245 in each group). This suggeststhat the consistent impairment in the RAM is not accounted for by theincreased time to complete the task taken by TG mice. Results in the Ymaze were also significantly different for TG and nTG mice (Groupeffect: p=0.011). Validation studies performed on non-transgenic miceindicate that the Y maze is a less sensitive measure than the RAM.

[0288] Measures of strength (INP) and activity indicate no differencesbetween TG and nTG mice. These are considered very rough measures, withonly large differences being detectable. There was, however, a decreasein the activity score for all mice over time (Age effect: p=0.070).There was a difference in body weight, with TG weighing 8% less thancontrols (Group effect: p=0.0003), primarily in female TG mice. However,this does not seem to have an effect on the results, as shown by thelack of any difference in strength (see above) or swimming speed (seebelow) between TG and nTG mice.

[0289] Results of the hidden platform task, considered here a test ofreference memory, show a consistent difference between TG and nTG mice.ANOVA reveals that the effects of transgenic status (Group effect:p=0.00016) and trial blocks (Block effect: p<0.00001) are significant.The effect of transgenic status on performance is accounted for byslower performance by TG mice across all trial blocks and ages. Analysisof Trial 1 reveals an effect of transgenic status (Group effect:p=0.018), suggesting a difference in performance before learning hasoccurred. However, an analysis of covariance, with trial 1 as thecovariate, still yields a significant deficit in TG mice (p=0.00051).

[0290] It was also possible that some physical differences between TGand nTG mice, rather than cognitive differences, could have beenresponsible for some of the performance differences seen in the watermaze tasks. However, no significant difference in strength or activitywas observed (see above). Another possibility considered was the effectof swimming speed on performance since a slower swimmer with equivalentcognitive ability would take longer to reach the platform. To test this,video tracking was used in the hidden platform task to measure thedistance travelled to reach the platform (a measure of the amount ofsearching done by the mice which is related to cognitive ability), theswimming speed (a measure of physical ability unrelated to cognitiveability), and the amount of time need to find the platform (a measure ofthe combination of both the distance travelled and the swimming speed).This was done in older and younger mice than reported above, using threetrials per block and no pretraining. The time needed to find theplatform was significantly different in TG and nTG mice (Group effect:p<0.0005), with the TG mice taking longer. However, the swimming speedwas not significantly different between TG and nTG mice (Group effect:p=0.879). Thus, the difference in time needed to find the platform islikely to be due to a cognitive difference between TG and nTG mice. Thisis confirmed by measures of the distance travelled to find the platform.The TG mice travelled significantly further than the nTG mice beforereaching the platform (Group effect: p<0.0005). These results indicatethat the differences seen between TG and nTG mice in the time to reachthe platform in the water maze tasks are due to differences in cognitiveability.

[0291] To test whether nTG mice retain a better memory of the platformlocation than TG mice, a probe trial was given immediately followinghidden platform training in which the platform was removed. Videotracking was used to determine the number of crossings of the formerplatform location made by the mice relative to crossings of non-platformlocations. There was a significant difference seen between the relativecrossings of TG and nTG mice (Group effect: p=0.006). This is evidencethat the nTG mice remember the former location of the platform betterthan TG mice.

[0292] It was also possible that the difference observed between TG andnTG mice in the time needed to reach the platform could have beeninfluenced by differences in perception of the cues or motivationaldifferences. To test this, TG and nTG mice were subjected to visibleplatform tasks in the water maze. For these tasks, a platform was placedin the pool so that it was visible above the water. Three differentplatforms were tested, a dark platform 25 mm above the surface (mostvisible), a gray platform 25 mm above the surface, and a dark platform 5mm above the surface (both less visible). The results show no differencein the time to find the most visible platform between TG and nTG mice(Group effect: p=0.403). There was not any greater decrease inperformance in TG mice when less visible platforms were used, suggestingthat their vision was as good as nTG mice. These results indicate thatperceptual and motivational differences do not influence the time toreach the platform in the water maze tasks described above.

[0293] Performance differences between TG and nTG mice were shown forRAM, Y maze, and water maze cognitive tasks in mice aged 4 to 8 months(2 to 12 months for the water maze). All of these differences indicate,and are consistent with, cognitive deficits in the transgenic mice as agroup. The various tasks combined to test working memory and referencememory, both of which are implicated in cognitive impairment observed inAlzheimer's victims. Example 10: Detection and Measurement ofAlzheimer's Disease Markers. A. Detection and Measurement of GFAP.

[0294] Glial fibrillary acidic protein (GFAP), a marker which increasesin AD brain tissue, was measured in the following manner. Tissueextracts were prepared from hippocampi of control and PDAPP transgenicmice, as described in Example 6, aged 14 months. Tissue was sonicated in10 volumes (v/w) of 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1 mMPMSF, 10 μg/ml leupeptin, 5 μg/ml calpain inhibitor 1. Proteindeterminations were made on the extract and SDS-PAGE sample buffer addedbefore boiling the samples for 5 minutes. SDS-PAGE was performed using12.5 μg of protein of each sample loaded onto 10% Tris-glycine gels(Novex). The proteins were transferred to Pro-Blot PVDF membranes bystandard methods. GFAP immunoreactive proteins were detected using ananti-GFAP antibody from Sigma (G9269) used at a dilution of 1:2,000. Anincrease in immunoreactivity in general was observed, and a smalleranti-GFAP reactive species was also found to increase substantially, inthe transgenic animals. In non-transgenic animals, this approximately 40kD fragment gave a mean densitometer signal of 142.47, while in thetransgenic animals, it gave a mean densitometer signal of 591.51. Thisdifference was significant, with a P value of 0.0286.

[0295] B. Detection of Gliosis.

[0296] Gliosis is one of the changes that is associated with theneuropathology of Alzheimer's disease. The isoquinoline carboxamide PK11195 has been shown to be a preferential marker of the peripheralbenzodiazepine sites associated with gliosis. These sites have beenshown to be enhanced in several diseases and animal models associatedwith neuronal damage and activated necroglia including stroke(Stephenson et al., J. Neuroscience 15:5263-5274 (1991)) and Alzheimer'sdisease (Diorio et al., Neurobiology of Aging 12:255-258 (1991)). Inparticular Diorio and colleagues have shown an approximate 200% increasein [³H] PK 11195 binding in some brain regions of AD patients, such asthe temporal cortex compared with age-matched controls. In this example,the brains from the PDAPP mouse, described in Example 6, were examinedfor qualitative and quantitative changes in the binding of [³H] PK 11195in order to correlate with the previously described AD diseasepathology. Two different approaches were utilized; radioreceptor bindingto homogenates of different brain regions and receptor autoradiography.

[0297] 1. Methods.

[0298] For the homogenate binding studies, PDAPP mice were euthanised bycervical dislocation and the brains rapidly dissected on ice.Homogenates (10 mg/ml wet weight) of cerebral cortex, hippocampus andcerebellum were prepared in 50 mM Tris HCl, pH 7.4. 0.3, 1.0 and 3.0 nM[³H] PK 11195 was incubated with these brain regions for 60 minutes at23° C. followed by rapid filtration over Whatman GF/B filters using aBrandell cell harvester. Non-specific binding was determined using 1 μMunlabelled PK 11195. Quantitation was performed by liquid scintillationspectrometry.

[0299] In the autoradiographic studies, PDAPP mice were euthanised usingcarbon dioxide, the brains removed and snap frozen in methyl butane/dryice. The brains were sectioned in the coronal plane through thehippocampus. Twenty micron thick sections were mounted on glass slidesand stored at −20° C. Sections were incubated at 1 hour at 23° C. in 170mM Tris-HCl, pH 7.4 containing 1 nM [³H] PK 11195. Non-specific bindingwas determined using 1 μM unlabelled PK 11195. Incubations wereterminated by rinsing sections twice for 5 minutes in ice-coldincubation buffer followed by a brief wash in ice-cold distilled water.Following rapid drying, sections were exposed to tritium Hyperfilm(Amersham International) for up to 5 weeks.

[0300] 2. Results.

[0301] Four heterozygous transgenic mice 3-4 months of age wereevaluated in the homogenate binding studies and compared withlitter-mate controls. No significant differences were observed betweenany of the brain regions of the transgenic animals and their respectivecontrols. [³H] PK 1119 autoradiography was performed to compare bindingin a 12-month old heterozygotic transgenic mouse with an aged-matchednon-transgenic control. Preliminary results from an autoradiogramexposed for five weeks indicated that several plaque-like structureswere labeled in the retrosplenial cortex of the transgenic mouse, aregion that invariably contains Aβ deposits. The pattern of labelingcorresponded to microglial cell or astrocytic clumps associated withplaques, rather than the more widespread pattern of astrocytosis ormicrogliosis in the hippocampal and cortical parenchyma. Thenon-transgenic mouse did not show this labeling pattern.

[0302] No changes were observed in the 3-4 month animals but someevidence for an increase in [³H] PK 11195 binding was seen in the12-month animal.

[0303] C. Detection and Measurement of Cholinergic Nerve Terminals.

[0304] A population of cholinergic neurones projecting to the forebrainhave been shown to be selectively decreased in the postmortem brains ofpatients diagnosed with Alzheimer's disease. Hemicholinium-3 is a potentinhibitor of high affinity choline uptake and has been shown to be agood marker of cholinergic nerve terminals (Pascual et al., J Neurochem54:792-800 (1990)). The total number of high affinity choline uptakesites in PDAPP transgenic animals, which are described in Example 6, hasbeen measured using both crude whole-brain preparations and homogenatesfrom selective brain regions using the selective ligand[³H]-Hemicholinium-3 ([³H]HCh-3). [³H]-Hemicholinium-3 binding wasdetermined using a modification of the methods described in Pascual etal. Mice were euthanised by asphyxiation with carbon dioxide and thebrains rapidly removed and dissected on ice. The cortex, cerebellum,striatum and hippocampus were homogenized in 5 ml of 10 mM phosphatebuffer without NaCl. Samples were spun at 17,000× g for 10 minutes, andthe pellets washed twice in 5 ml 10 mM PO₄ buffer. The final pellet wasresuspended in 5 ml 1× phosphate buffered saline (PBS) to produce aprotein concentration of 0.5 mg/ml. Brain regions were assayed intriplicate for high affinity choline uptake sites by the addition of[³H]HCh-3 (3 nM final concentration). Following a 20 minute incubation,assays were terminated by rapid filtration through Whatman GF/B filtersusing a Brandell cell harvester and washing with PBS. Filters weretransferred into scintillation vials, and specific binding estimated byliquid scintillation spectrometry.

[0305] D. Detection and Measurement of Sodium-Potassium ATPase.

[0306] Ouabain has been shown to bind specifically to high affinitysites in mammalian brain and that these sites correspond to a neuronalform of sodium-potassium ATPase (Na/K-ATPase; Hauger et al., J Neurochem44:1709-1715 (1985)). These sites have been shown to decrease in animalmodels of neurodegenerative diseases. Alzheimer's disease ischaracterized by massive neurodegeneration (DeLacoste and White,Neurobiology of Aging 4(1):1-16 (1993)).

[0307] In order to estimate the extent of neurodegeneration in the PDAPPmouse the binding of ouabain was determined in mouse brain homogenates.Methods were adapted from those described by Hauger et al. Brain tissuewas homogenized in 100 mM Tris HCl, pH 7.4 containing 200 mM NaCl and 10mM MgCl₂ and resuspended in assay buffer to produce a finalconcentration of 100 μg protein per assay. Specific binding wasdetermined with 1 to 200 nM [³H] ouabain in a solution of 5 mM ATP, 100mM Tris HCl, pH 7.4, 10 mM MgCl₂, and 200 mM NaCl, incubated for 30minutes at 37° C. Non specific binding was determined in the presence of100 mM ouabain and the absence of ATP. Assays were terminated by rapidfiltration over Whatman GF/B filters. Tubes were washed with ice cold 50mM Tris HCl, pH 7.4, 15 mM KCl, 5 mM MgCl₂. Filters were transferredinto scintillation vials, and specific binding estimated by liquidscintillation spectrometry.

[0308] Measurements of Na/K-ATPase and Mg-ATPase activity in braintissue of PDAPP transgenic mice of various ages and in non-transgeniccontrol brain tissue. These results show some significant differences inactivity between transgenic and non-transgenic samples in older mice.Mouse brain homogenates from 4, 8, and 12 month old PDAPP transgenic(TG) mice, and from non-transgenic (nTG) mice, were prepared and assayedgenerally as described above. The activity of Mg-ATPase was alsodetermined. The results are shown in Tables 7 and 8. TABLE 7 Na/K-ATPaseActivity in PDAPP Transgenic Mouse Brain. Na/K-ATPase rate Age Tissue(pmole Pi/mg protein/min) % of nTG 4 TG hippocampus 2.57 ± 0.62  88 ± 144 nTG hippocampus 2.53 ± 0.51 — 4 TG cortex 0.57 ± 0.13 72 ± 9 4 nTGcortex 0.77 ± 0.17 — 4 TG cerebellum 1.39 ± 0.17  85 ± 14 4 nTGcerebellum 2.14 ± 0.53 — 8 TG hippocampus 4.63 ± 1.72 153 ± 53 8 nTGhippocampus 2.87 ± 0.41 — 8 TG cortex 1.16 ± 0.08 121 ± 23 8 nTG cortex1.15 ± 0.20 — 8 TG cerebellum 2.74 ± 0.81 183 ± 53 8 nTG cerebellum 1.47± 0.13 — 12 TG hippocampus 1.66 ± 0.36 58 ± 9 12 nTG hippocampus 3.11 ±0.94 — 12 TG cortex 1.45 ± 0.40 109 ± 17 12 nTG cortex 1.60 ± 0.46 — 12TG cerebellum 1.43 ± 0.32 74 ± 7 12 nTG cerebellum 2.04 ± 0.64 —

[0309] TABLE 8 Mg-ATPase Activity in PDAPP Transgenic Mouse Brain.Mg-ATPase rate Age Tissue (pmole Pi/mg protein/min) % of nTG 4 TGhippocampus 2.83 ± 0.38 110 ± 14 4 nTG hippocampus 2.44 ± 0.54 — 4 TGcortex 1.74 ± 0.14 92 ± 5 4 nTG cortex 1.87 ± 0.18 — 4 TG cerebellum2.58 ± 0.44 100 ± 12 4 nTG cerebellum 2.59 ± 0.35 — 8 TG hippocampus3.28 ± 0.69  99 ± 22 8 nTG hippocampus 3.32 ± 0.39 — 8 TG cortex 1.72 ±0.11 73 ± 6 8 nTG cortex 2.40 ± 0.31 — 8 TG cerebellum 3.19 ± 0.49 113 ±15 8 nTG cerebellum 2.77 ± 0.23 — 12 TG hippocampus 1.48 ± 0.21 65 ± 712 nTG hippocampus 2.33 ± 0.46 — 12 TG cortex 1.60 ± 0.30 76 ± 7 12 nTGcortex 2.06 ± 0.40 — 12 TG cerebellum 1.61 ± 0.26 78 ± 8 12 nTGcerebellum 1.93 ± 0.99 —

[0310] The difference in Na/K-ATPase activity between transgenic andnon-transgenic tissue is significant (p<0.05) in the case of 12 monthold cerebellum, and is highly significant (p<0.01) in the case of 12month old hippocampus. The difference in Mg-ATPase activity betweentransgenic and non-transgenic tissue is significant (p<0.05) in the caseof 8 and 12 month old cortex, and is highly significant (p<0.01) in thecase of 12 month old hippocampus.

[0311] E. In situ Hybridization With Probes to Neurotrophic Factors.

[0312] The use of in situ hybridization to detect and localize mRNAs forspecific gene products is well documented in the literature (Lewis etal., Molecular Imaging in Neuroscience: A Practical Approach (New York,Oxford University Press. 1st ed., 1-21, 1993), Lu and Gillett, CellVision 1(2):169-176 (1994), Sirinathsinghji and Dunnett, MolecularImaging in Neuroscience: A Practical Approach (New York, OxfordUniversity Press. 1st., ed. 43-67, 1993), Lawerence and Singer, Nuc.Acids Res. 13:1777-1799 (1985), Zeller and Rogers, Current Protocols inMolecular Biology (New York, John Wiley and Sons. 14.3.1-14.5.5, 1995)).For illustrative purposes, the specific example described below utilizes³⁵S-radiolabeled oligodeoxyribonucleotide probes to detect BDNF mRNA incryostat sectioned mouse brain from the PDAPP transgenic mouse describedin Example 6 and non-transgenic control mice. However, ³³P-labeledradioactive DNA probes, as well as in vitro transcribed complementaryRNA probes, could be used as well. Non radioactive probe labelingmethods may also be used (Knoll, Current Protocols in Molecular Biology(New York, John Wiley and Sons. 14.7.1-14.7.14, 1995)). Additionally,the choice of tissue pre-treatment for hybridization with probe (forexample, paraffin embedded sections) and post-hybridization washesdepend on the method used, examples of which are described in thereferences cited above. Known and appropriate precautions against RNasecontamination should be employed and are also discussed in the abovereferences.

[0313] 1. Tissue Preparation.

[0314] Freshly dissected whole brains, or sub-regions of interest, fromtransgenic or control mice at various developmental stages, orpost-natal ages, are snap frozen in isopentane pre-equilibrated to −70°C. If desired, the animals may be perfused with PBS to eliminatecirculating cells from brain prior to dissection. The brains are removedfollowing 15 to 20 seconds immersion in isopentane, wrapped in aluminumfoil, labeled appropriately, and stored at −80° C. for sectioning. Itshould be noted that although the signal from in situ hybridization tocryostat sectioned tissues is more sensitive than to paraffin embeddedsections, it is dependent upon the time from dissection tohybridization. Frozen tissue is preferably analyzed by hybridizationwith probe within six weeks. RNA integrity in tissues declines beyondthis time. Thus, if longer time periods between dissection and analysisare anticipated, the tissue should be fixed (see, for example, Lu andGillett) before long term storage at −80° C.

[0315] Prior to sectioning, Probe-On-Plus glass slides (FisherScientific, Pittsburgh, Pa.) can be made RNAase free by overnightsoaking in absolute ethanol, air dried briefly in a dust freeenvironment, and baked at 180° C. for a minimum of 4 hours. Aftercooling to room temperature, the slides are coated with 0.01%poly-lysine (prepared in DEPC treated H₂O) for approximately 5 seconds,and air dried in a dust free area. The coated slides can be stored forup to one month before use in a slide box with silica gel or drieritepellets.

[0316] For sectioning, the frozen brain stored at −80° C. is transferredto a cryostat at −20° C., mounted onto a sectioning block, embedded inOCT.®, and allowed to equilibrate. The tissue is then cut into 7 to 14μm thick sections using a sterilized microtome knife (treated with 70%EtOH in DEPC H₂O), and thaw mounted onto poly-lysine coated slides. Theslides are kept at −20° C. until the sectioning is complete. Thesections are fixed and dehydrated by immersing the slides sequentiallyin the solutions noted below. 1. once in 4% paraformaldehyde, 1X PBS, pH7.4, at 0° C. for 5 minutes (this solution should be made fresh, and canbe stored for up to 1 week at 4° C.); 2. twice in 1X PBS, 2.5 minuteseach time; 3. once in 50% EtOH in DEPC H₂O for 5 minutes; 4. once in 70%EtOH in DEPC H₂O for 5 minutes; 5. once in 95% EtOH in DEPC H₂O for 5minutes;

[0317] The fixed sections are stored immersed in the 95% EtOH/DEPC H₂Osolution at 4° C. until use. If the sections are not fixed immediately,they may be stored at −80° C. in the presence of drierite until use. Inthis case the sections are allowed to equilibrate to room temperatureprior to the fixation/dehydration steps.

[0318] 2. Probe Design and Preparation.

[0319] The sequence of the mouse BDNF mRNA/cDNA (accession #55573) isavailable from the Genbank database of Nucleic acid sequences (NCBI,Bethesda, Md.). Anti-sense oligodeoxynucleotide probes against BDNF weredesigned using the primer select module of the DNAstar™ software package(Lasergene Inc., Madison, Wis.). Numerous other software packages, suchOligo® (NBI, Plymouth, Minn.), offer similar capabilities, and are alsosuitable. Candidate probes of 45 to 55 nucleotides length, approximately50% G+C content, and hybridizing to the pre-cursor or mature peptideencoding regions of the BDNF mRNA were synthesized on an ABI 380B DNAsynthesizer. As specificity controls, sense oligonucleotidescorresponding to each probe were also synthesized. Using the conventionthat the first nucleotide in the BDNF coding region is position 1, theBDNF probes synthesized correspond to BDNF nucleotide positions 47 to 94(probes 2710 & 2711), 158 to 203 (probes 2712 & 2713), 576 to 624(probes 2714 & 2715), and 644 to 692 (probes 2716 & 2717). The evennumbered oligonucleotides are probes for the sense strand, and the oddnumbered oligonucleotides are probes for the anti-sense strand.

[0320] For radiolabeling, the probes are gel purified on denaturingacrylamide gels and reconstituted in H₂O using standard protocols(Sambrook et al.). The probes (30 to 35 ng, 2 pmoles) are labeled by 3′homopolymeric tailing using terminal deoxynucleotidyl transferase(Promega, Madison Wis.) and ³⁵S-dATP (1000 Ci/mmol, Amersham Inc.)according to the enzyme manufacturer's recommendation. The radiolabeledprobes are purified by column chromatography on size exclusion mini-spincolumns (Biospin-6, Biorad Inc., Hercules, Calif.). The specificactivity of the probes is quantitated by scintillation counting. Typicalspecific activities of the probes ranged from 1×10⁹ to 5×10⁹ cpm/μg.

[0321] 3. Tissue Hybridization and Post Hybridization Washes.

[0322] In preparation for hybridization, the desired number of slidesare removed from storage under alcohol, and allowed to air drythoroughly in the slide rack (approximately 1 hour). Meanwhile, theprobe is heat denatured in a boiling H₂O bath for 2 to 5 minutes, quickchilled in an ice/H₂O bath, and diluted in hybridization buffer (10%dextran sulfate, 50% deionized formamide, 4× SSC, 5× Dehardts, 100 μg/mlsheared salmon sperm DNA, 100 μg/ml polyadenylic acid) to a finalconcentration of 5×10³ to 10×10³ cpm/μl. DTT is added to a 10 mM finalconcentration.

[0323] For hybridization, 100 μl of diluted probe in hybridizationbuffer (corresponding to 0.5×10⁶ to 1.0×10⁶ cpm probe) is carefullyapplied to each section being hybridized with probe. The solution isgently spread over the section with a pipet tip to cover the entiresection(s) on each slide. The slides containing probe are then placed inhumidified hybridization chambers at 42° C. for hybridization overnightwith the probe. The hybridization chambers can be covered utility boxes,or acrylic boxes, with raised platforms to accommodate slides. The boxesare lined with filter paper (or paper towels), saturated in 4× SSC, 50%formamide, and humidified by pre-incubating them with closed lids in a42° C. incubator for 1 to 3 hours before the slides are placed insidethem. Although cover slips can be placed on the sections after thehybridization buffer is applied, this is not necessary provided thehybridization chambers are adequately humidified during the procedure.If pre-hybridization is used to obtain a lower background, the sectionsmay be incubated at 42° C. under 50 μl hybridization buffer (minusprobe) per section for 1 to 2 hours. After this time, an equal volume ofhybridization buffer containing probe at twice the concentrationdescribed above is applied to each section, and hybridization is carriedout as described above.

[0324] For washes, the slides are transferred from the hybridizationchamber to a slide holder. The slides can be placed in the slide holderevery 4th or 5th slot so as to allow adequate flow of wash solution overthe surface of each section. This placement can significantly lowersbackground on the sections. A moderate flow rate of wash solution overthe surface of the sections promotes removal of unhybridized probe, andconsequently reduces background. This is best accomplished during the55° C. wash steps by suspending the slides in the slide holder, above amagnetic stir bar. The stir bar is preferably placed approximately oneinch under the slide holder. This can be done by hanging the slideholder(s) from pipets straddling the wash chamber. A large beaker, or a4 to 6 inch deep Pyrex baking dish makes a suitable wash chamber. Thewash chamber is placed on a hot-plate stirrer, the temperature settingof which is precalibrated to maintain the wash solution at 55° C. duringthe procedure. The changes of wash solution are made usingpre-equilibrated solution. Washes and post wash dehydration of thesections are carried out as follows: 1. twice in 1X SSC at roomtemperature for 5 minutes each time; 2. three times in 1X SSC at 55° C.for 30 minutes each time; 3. once in 1X SSC at room temperature for 1min. 4. once in 0.1X SSC at room temperature for 15 seconds; 5. once inultra pure H₂O at room temperature for 15 seconds; 6. once in 50% EtOHfor 15 seconds; 7. once in 70% EtOH for 15 seconds; 8. once in 95% EtOHfor 15 seconds;

[0325] The sections are air dried thoroughly at room temperature for 2hours, followed by 30 minutes at 55° C. The dried sections on the slidesare placed in autoradiographic cassette and exposed to X-ray film(Hyperfilm, β-max, Amersham Inc., Arlington Heights, Ill.) at 4° C. for2 to 3 days to estimate the exposure time required under emulsion. TheX-ray film is developed according to the manufacturers recommendation.The sections are coated with emulsion (Amersham LM-1, #RPN40) by dippingin emulsion at 42° C. under appropriate safelight conditions. Theemulsion coated slides are air dried on a cooled surface forapproximately 30 minutes, and transferred to a plastic slide boxcontaining a drying agent (drierite pellets). The seams of the box canbe sealed with black tape, and the box wrapped in several layers ofaluminum foil to ensure a light-tight enclosure. Following 2 to 4 hoursat room temperature to finish the drying, the boxes are transferred to4° C. for autoradiographic exposure for 2 to 6 weeks. Prior todeveloping the emulsion coated slides, the box is removed from therefrigerator and allowed to equilibrate to room temperature forapproximately 1 hour. The slides are then developed according to themanufacturers instructions, air dried for 1 to 2 hours, and if desired,counterstained with the appropriate counterstain.

[0326] Modifications and variations of the making and testing oftransgenic animal models for testing of Alzheimer's disease will beobvious to those skilled in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

1 10 2085 base pairs nucleic acid double linear cDNA NO NO CDS 1-2085/function= “coding region for APP695.” 1 ATG CTG CCC GGT TTG GCA CTG CTCCTG CTG GCC GCC TGG ACG GCT CGG 48 Met Leu Pro Gly Leu Ala Leu Leu LeuLeu Ala Ala Trp Thr Ala Arg 1 5 10 15 GCG CTG GAG GTA CCC ACT GAT GGTAAT GCT GGC CTG CTG GCT GAA CCC 96 Ala Leu Glu Val Pro Thr Asp Gly AsnAla Gly Leu Leu Ala Glu Pro 20 25 30 CAG ATT GCC ATG TTC TGT GGC AGA CTGAAC ATG CAC ATG AAT GTC CAG 144 Gln Ile Ala Met Phe Cys Gly Arg Leu AsnMet His Met Asn Val Gln 35 40 45 AAT GGG AAG TGG GAT TCA GAT CCA TCA GGGACC AAA ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly ThrLys Thr Cys Ile Asp 50 55 60 ACC AAG GAA GGC ATC CTG CAG TAT TGC CAA GAAGTC TAC CCT GAA CTG 240 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu ValTyr Pro Glu Leu 65 70 75 80 CAG ATC ACC AAT GTG GTA GAA GCC AAC CAA CCAGTG ACC ATC CAG AAC 288 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro ValThr Ile Gln Asn 85 90 95 TGG TGC AAG CGG GGC CGC AAG CAG TGC AAG ACC CATCCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His ProHis Phe Val 100 105 110 ATT CCC TAC CGC TGC TTA GTT GGT GAG TTT GTA AGTGAT GCC CTT CTC 384 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser AspAla Leu Leu 115 120 125 GTT CCT GAC AAG TGC AAA TTC TTA CAC CAG GAG AGGATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg MetAsp Val Cys 130 135 140 GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC AAA GAGACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu ThrCys Ser Glu 145 150 155 160 AAG AGT ACC AAC TTG CAT GAC TAC GGC ATG TTGCTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu LeuPro Cys Gly Ile 165 170 175 GAC AAG TTC CGA GGG GTA GAG TTT GTG TGT TGCCCA CTG GCT GAA GAA 576 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys ProLeu Ala Glu Glu 180 185 190 AGT GAC AAT GTG GAT TCT GCT GAT GCG GAG GAGGAT GAC TCG GAT GTC 624 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu AspAsp Ser Asp Val 195 200 205 TGG TGG GGC GGA GCA GAC ACA GAC TAT GCA GATGGG AGT GAA GAC AAA 672 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp GlySer Glu Asp Lys 210 215 220 GTA GTA GAA GTA GCA GAG GAG GAA GAA GTG GCTGAG GTG GAA GAA GAA 720 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala GluVal Glu Glu Glu 225 230 235 240 GAA GCC GAT GAT GAC GAG GAC GAT GAG GATGGT GAT GAG GTA GAG GAA 768 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp GlyAsp Glu Val Glu Glu 245 250 255 GAG GCT GAG GAA CCC TAC GAA GAA GCC ACAGAG AGA ACC ACC AGC ATT 816 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr GluArg Thr Thr Ser Ile 260 265 270 GCC ACC ACC ACC ACC ACC ACC ACA GAG TCTGTG GAA GAG GTG GTT CGA 864 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser ValGlu Glu Val Val Arg 275 280 285 GTT CCT ACA ACA GCA GCC AGT ACC CCT GATGCC GTT GAC AAG TAT CTC 912 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp AlaVal Asp Lys Tyr Leu 290 295 300 GAG ACA CCT GGG GAT GAG AAT GAA CAT GCCCAT TTC CAG AAA GCC AAA 960 Glu Thr Pro Gly Asp Glu Asn Glu His Ala HisPhe Gln Lys Ala Lys 305 310 315 320 GAG AGG CTT GAG GCC AAG CAC CGA GAGAGA ATG TCC CAG GTC ATG AGA 1008 Glu Arg Leu Glu Ala Lys His Arg Glu ArgMet Ser Gln Val Met Arg 325 330 335 GAA TGG GAA GAG GCA GAA CGT CAA GCAAAG AAC TTG CCT AAA GCT GAT 1056 Glu Trp Glu Glu Ala Glu Arg Gln Ala LysAsn Leu Pro Lys Ala Asp 340 345 350 AAG AAG GCA GTT ATC CAG CAT TTC CAGGAG AAA GTG GAA TCT TTG GAA 1104 Lys Lys Ala Val Ile Gln His Phe Gln GluLys Val Glu Ser Leu Glu 355 360 365 CAG GAA GCA GCC AAC GAG AGA CAG CAGCTG GTG GAG ACA CAC ATG GCC 1152 Gln Glu Ala Ala Asn Glu Arg Gln Gln LeuVal Glu Thr His Met Ala 370 375 380 AGA GTG GAA GCC ATG CTC AAT GAC CGCCGC CGC CTG GCC CTG GAG AAC 1200 Arg Val Glu Ala Met Leu Asn Asp Arg ArgArg Leu Ala Leu Glu Asn 385 390 395 400 TAC ATC ACC GCT CTG CAG GCT GTTCCT CCT CGG CCT CGT CAC GTG TTC 1248 Tyr Ile Thr Ala Leu Gln Ala Val ProPro Arg Pro Arg His Val Phe 405 410 415 AAT ATG CTA AAG AAG TAT GTC CGCGCA GAA CAG AAG GAC AGA CAG CAC 1296 Asn Met Leu Lys Lys Tyr Val Arg AlaGlu Gln Lys Asp Arg Gln His 420 425 430 ACC CTA AAG CAT TTC GAG CAT GTGCGC ATG GTG GAT CCC AAG AAA GCC 1344 Thr Leu Lys His Phe Glu His Val ArgMet Val Asp Pro Lys Lys Ala 435 440 445 GCT CAG ATC CGG TCC CAG GTT ATGACA CAC CTC CGT GTG ATT TAT GAG 1392 Ala Gln Ile Arg Ser Gln Val Met ThrHis Leu Arg Val Ile Tyr Glu 450 455 460 CGC ATG AAT CAG TCT CTC TCC CTGCTC TAC AAC GTG CCT GCA GTG GCC 1440 Arg Met Asn Gln Ser Leu Ser Leu LeuTyr Asn Val Pro Ala Val Ala 465 470 475 480 GAG GAG ATT CAG GAT GAA GTTGAT GAG CTG CTT CAG AAA GAG CAA AAC 1488 Glu Glu Ile Gln Asp Glu Val AspGlu Leu Leu Gln Lys Glu Gln Asn 485 490 495 TAT TCA GAT GAC GTC TTG GCCAAC ATG ATT AGT GAA CCA AGG ATC AGT 1536 Tyr Ser Asp Asp Val Leu Ala AsnMet Ile Ser Glu Pro Arg Ile Ser 500 505 510 TAC GGA AAC GAT GCT CTC ATGCCA TCT TTG ACC GAA ACG AAA ACC ACC 1584 Tyr Gly Asn Asp Ala Leu Met ProSer Leu Thr Glu Thr Lys Thr Thr 515 520 525 GTG GAG CTC CTT CCC AGC CTGGAC GAT CTC CAG CCG TGG CAT TCT TTT 1632 Val Glu Leu Leu Pro Val Asn GlyGlu Phe Ser Leu Asp Asp Leu Gln 530 535 540 GTG AAT GGA GAG TTC GGG GCTGAC TCT GTG CCA GCC AAC ACA GAA AAC 1680 Pro Trp His Ser Phe Gly Ala AspSer Val Pro Ala Asn Thr Glu Asn 545 550 555 560 GAA GTT GAG CCT GTT GATGCC CGC CCT GCT GCC GAC CGA GGA CTG ACC 1728 Glu Val Glu Pro Val Asp AlaArg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 ACT CGA CCA GGT TCT GGGTTG ACA AAT ATC AAG ACG GAG GAG ATC TCT 1776 Thr Arg Pro Gly Ser Gly LeuThr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 GAA GTG AAG ATG GAT GCAGAA TTC CGA CAT GAC TCA GGA TAT GAA GTT 1824 Glu Val Lys Met Asp Ala GluPhe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 CAT CAT CAA AAA TTG GTGTTC TTT GCA GAA GAT GTG GGT TCA AAC AAA 1872 His His Gln Lys Leu Val PhePhe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 GGT GCA ATC ATT GGA CTCATG GTG GGC GGT GTT GTC ATA GCG ACA GTG 1920 Gly Ala Ile Ile Gly Leu MetVal Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 ATC GTC ATC ACC TTGGTG ATG CTG AAG AAG AAA CAG TAC ACA TCC ATT 1968 Ile Val Ile Thr Leu ValMet Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 CAT CAT GGT GTG GTGGAG GTT GAC GCC GCT GTC ACC CCA GAG GAG CGC 2016 His His Gly Val Val GluVal Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 CAC CTG TCC AAG ATGCAG CAG AAC GGC TAC GAA AAT CCA ACC TAC AAG 2064 His Leu Ser Lys Met GlnGln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 TTC TTT GAG CAG ATGCAG AAC 2085 Phe Phe Glu Gln Met Gln Asn 690 695 695 amino acids aminoacid linear protein 2 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala AlaTrp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala GlyLeu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn MetHis Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly ThrLys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln GluVal Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn GlnPro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys LysThr His Pro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly GluPhe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe LeuHis Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp HisThr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn LeuHis Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe ArgGly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp AsnVal Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp TrpGly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 ValVal Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val ValArg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp LysTyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe GlnLys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg MetSer Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala LysAsn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe GlnGlu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg GlnGln Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu AsnAsp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala LeuGln Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu LysLys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu LysHis Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala GlnIle Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 ArgMet Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys ThrThr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp AspLeu Gln 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala AsnThr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala AlaAsp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn IleLys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe ArgHis Asp Ser Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu Val Phe PheAla Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu MetVal Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr LeuVal Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly ValVal Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu SerLys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe PheGlu Gln Met Gln Asn 690 695 2253 base pairs nucleic acid double linearcDNA NO NO CDS 1-2253 /function= “coding region for APP751.” 3 ATG CTGCCC GGT TTG GCA CTG CTC CTG CTG GCC GCC TGG ACG GCT CGG 48 Met Leu ProGly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 GCG CTGGAG GTA CCC ACT GAT GGT AAT GCT GGC CTG CTG GCT GAA CCC 96 Ala Leu GluVal Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 CAG ATT GCCATG TTC TGT GGC AGA CTG AAC ATG CAC ATG AAT GTC CAG 144 Gln Ile Ala MetPhe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 AAT GGG AAG TGGGAT TCA GAT CCA TCA GGG ACC AAA ACC TGC ATT GAT 192 Asn Gly Lys Trp AspSer Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 ACC AAG GAA GGC ATCCTG CAG TAT TGC CAA GAA GTC TAC CCT GAA CTG 240 Thr Lys Glu Gly Ile LeuGln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 CAG ATC ACC AAT GTGGTA GAA GCC AAC CAA CCA GTG ACC ATC CAG AAC 288 Gln Ile Thr Asn Val ValGlu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 TGG TGC AAG CGG GGC CGCAAG CAG TGC AAG ACC CAT CCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arg LysGln Cys Lys Thr His Pro His Phe Val 100 105 110 ATT CCC TAC CGC TGC TTAGTT GGT GAG TTT GTA AGT GAT GCC CTT CTC 384 Ile Pro Tyr Arg Cys Leu ValGly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 GTT CCT GAC AAG TGC AAATTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys PheLeu His Gln Glu Arg Met Asp Val Cys 130 135 140 GAA ACT CAT CTT CAC TGGCAC ACC GTC GCC AAA GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp HisThr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 AAG AGT ACC AAC TTGCAT GAC TAC GGC ATG TTG CTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu HisAsp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 GAC AAG TTC CGA GGGGTA GAG TTT GTG TGT TGC CCA CTG GCT GAA GAA 576 Asp Lys Phe Arg Gly ValGlu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 AGT GAC AAT GTG GATTCT GCT GAT GCG GAG GAG GAT GAC TCG GAT GTC 624 Ser Asp Asn Val Asp SerAla Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 TGG TGG GGC GGA GCAGAC ACA GAC TAT GCA GAT GGG AGT GAA GAC AAA 672 Trp Trp Gly Gly Ala AspThr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 GTA GTA GAA GTA GCAGAG GAG GAA GAA GTG GCT GAG GTG GAA GAA GAA 720 Val Val Glu Val Ala GluGlu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 GAA GCC GAT GATGAC GAG GAC GAT GAG GAT GGT GAT GAG GTA GAG GAA 768 Glu Ala Asp Asp AspGlu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 GAG GCT GAG GAACCC TAC GAA GAA GCC ACA GAG AGA ACC ACC AGC ATT 816 Glu Ala Glu Glu ProTyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 GCC ACC ACC ACCACC ACC ACC ACA GAG TCT GTG GAA GAG GTG GTT CGA 864 Ala Thr Thr Thr ThrThr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 GAG GTG TGC TCTGAA CAA GCC GAG ACG GGG CCG TGC CGA GCA ATG ATC 912 Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 TCC CGC TGG TACTTT GAT GTG ACT GAA GGG AAG TGT GCC CCA TTC TTT 960 Ser Arg Trp Tyr PheAsp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 TAC GGC GGATGT GGC GGC AAC CGG AAC AAC TTT GAC ACA GAA GAG TAC 1008 Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 TGC ATG GCCGTG TGT GGC AGC GCC ATT CCT ACA ACA GCA GCC AGT ACC 1056 Cys Met Ala ValCys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350 CCT GAT GCCGTT GAC AAG TAT CTC GAG ACA CCT GGG GAT GAG AAT GAA 1104 Pro Asp Ala ValAsp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 CAT GCC CATTTC CAG AAA GCC AAA GAG AGG CTT GAG GCC AAG CAC CGA 1152 His Ala His PheGln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380 GAG AGA ATGTCC CAG GTC ATG AGA GAA TGG GAA GAG GCA GAA CGT CAA 1200 Glu Arg Met SerGln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln 385 390 395 400 GCA AAGAAC TTG CCT AAA GCT GAT AAG AAG GCA GTT ATC CAG CAT TTC 1248 Ala Lys AsnLeu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe 405 410 415 CAG GAGAAA GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC GAG AGA CAG 1296 Gln Glu LysVal Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln 420 425 430 CAG CTGGTG GAG ACA CAC ATG GCC AGA GTG GAA GCC ATG CTC AAT GAC 1344 Gln Leu ValGlu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445 CGC CGCCGC CTG GCC CTG GAG AAC TAC ATC ACC GCT CTG CAG GCT GTT 1392 Arg Arg ArgLeu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val 450 455 460 CCT CCTCGG CCT CGT CAC GTG TTC AAT ATG CTA AAG AAG TAT GTC CGC 1440 Pro Pro ArgPro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480 GCAGAA CAG AAG GAC AGA CAG CAC ACC CTA AAG CAT TTC GAG CAT GTG 1488 Ala GluGln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val 485 490 495 CGCATG GTG GAT CCC AAG AAA GCC GCT CAG ATC CGG TCC CAG GTT ATG 1536 Arg MetVal Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met 500 505 510 ACACAC CTC CGT GTG ATT TAT GAG CGC ATG AAT CAG TCT CTC TCC CTG 1584 Thr HisLeu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu 515 520 525 CTCTAC AAC GTG CCT GCA GTG GCC GAG GAG ATT CAG GAT GAA GTT GAT 1632 Leu TyrAsn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp 530 535 540 GAGCTG CTT CAG AAA GAG CAA AAC TAT TCA GAT GAC GTC TTG GCC AAC 1680 Glu LeuLeu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560ATG ATT AGT GAA CCA AGG ATC AGT TAC GGA AAC GAT GCT CTC ATG CCA 1728 MetIle Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575TCT TTG ACC GAA ACG AAA ACC ACC GTG GAG CTC CTT CCC GTG AAT GGA 1776 SerLeu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590GAG TTC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT GGG GCT GAC 1824 GluPhe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp 595 600 605TCT GTG CCA GCC AAC ACA GAA AAC GAA GTT GAG CCT GTT GAT GCC CGC 1872 SerVal Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620CCT GCT GCC GAC CGA GGA CTG ACC ACT CGA CCA GGT TCT GGG TTG ACA 1920 ProAla Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635640 AAT ATC AAG ACG GAG GAG ATC TCT GAA GTG AAG ATG GAT GCA GAA TTC 1968Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe 645 650655 CGA CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA TTG GTG TTC TTT 2016Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe 660 665670 GCA GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT GGA CTC ATG GTG 2064Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val 675 680685 GGC GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC TTG GTG ATG CTG 2112Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu 690 695700 AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC 2160Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp 705 710715 720 GCC GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG CAG CAG AAC2208 Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn 725730 735 GGC TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG CAG AAC 2253Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn 740 745 750751 amino acids amino acid linear protein 4 Met Leu Pro Gly Leu Ala LeuLeu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro ThrAsp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe CysGly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp SerAsp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile LeuGln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn ValVal Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg GlyArg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr ArgCys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro AspLys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu ThrHis Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu AspLys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val GluGlu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly AspGlu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr GluArg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu SerVal Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly GlyAsn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val CysGly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350 Pro Asp Ala ValAsp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 His Ala HisPhe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380 Glu ArgMet Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln 385 390 395 400Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe 405 410415 Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln 420425 430 Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp435 440 445 Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln AlaVal 450 455 460 Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys TyrVal Arg 465 470 475 480 Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys HisPhe Glu His Val 485 490 495 Arg Met Val Asp Pro Lys Lys Ala Ala Gln IleArg Ser Gln Val Met 500 505 510 Thr His Leu Arg Val Ile Tyr Glu Arg MetAsn Gln Ser Leu Ser Leu 515 520 525 Leu Tyr Asn Val Pro Ala Val Ala GluGlu Ile Gln Asp Glu Val Asp 530 535 540 Glu Leu Leu Gln Lys Glu Gln AsnTyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560 Met Ile Ser Glu Pro ArgIle Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575 Ser Leu Thr Glu ThrLys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590 Glu Phe Ser LeuAsp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp 595 600 605 Ser Val ProAla Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620 Pro AlaAla Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635 640Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe 645 650655 Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe 660665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val675 680 685 Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val MetLeu 690 695 700 Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val GluVal Asp 705 710 715 720 Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser LysMet Gln Gln Asn 725 730 735 Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe GluGln Met Gln Asn 740 745 750 2310 base pairs nucleic acid double linearcDNA NO NO CDS 1-2310 /function= “coding region for APP770.” 5 ATG CTGCCC GGT TTG GCA CTG CTC CTG CTG GCC GCC TGG ACG GCT CGG 48 Met Leu ProGly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 GCG CTGGAG GTA CCC ACT GAT GGT AAT GCT GGC CTG CTG GCT GAA CCC 96 Ala Leu GluVal Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 CAG ATT GCCATG TTC TGT GGC AGA CTG AAC ATG CAC ATG AAT GTC CAG 144 Gln Ile Ala MetPhe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 AAT GGG AAG TGGGAT TCA GAT CCA TCA GGG ACC AAA ACC TGC ATT GAT 192 Asn Gly Lys Trp AspSer Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 ACC AAG GAA GGC ATCCTG CAG TAT TGC CAA GAA GTC TAC CCT GAA CTG 240 Thr Lys Glu Gly Ile LeuGln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 CAG ATC ACC AAT GTGGTA GAA GCC AAC CAA CCA GTG ACC ATC CAG AAC 288 Gln Ile Thr Asn Val ValGlu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 TGG TGC AAG CGG GGC CGCAAG CAG TGC AAG ACC CAT CCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arg LysGln Cys Lys Thr His Pro His Phe Val 100 105 110 ATT CCC TAC CGC TGC TTAGTT GGT GAG TTT GTA AGT GAT GCC CTT CTC 384 Ile Pro Tyr Arg Cys Leu ValGly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 GTT CCT GAC AAG TGC AAATTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys PheLeu His Gln Glu Arg Met Asp Val Cys 130 135 140 GAA ACT CAT CTT CAC TGGCAC ACC GTC GCC AAA GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp HisThr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 AAG AGT ACC AAC TTGCAT GAC TAC GGC ATG TTG CTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu HisAsp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 GAC AAG TTC CGA GGGGTA GAG TTT GTG TGT TGC CCA CTG GCT GAA GAA 576 Asp Lys Phe Arg Gly ValGlu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 AGT GAC AAT GTG GATTCT GCT GAT GCG GAG GAG GAT GAC TCG GAT GTC 624 Ser Asp Asn Val Asp SerAla Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 TGG TGG GGC GGA GCAGAC ACA GAC TAT GCA GAT GGG AGT GAA GAC AAA 672 Trp Trp Gly Gly Ala AspThr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 GTA GTA GAA GTA GCAGAG GAG GAA GAA GTG GCT GAG GTG GAA GAA GAA 720 Val Val Glu Val Ala GluGlu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 GAA GCC GAT GATGAC GAG GAC GAT GAG GAT GGT GAT GAG GTA GAG GAA 768 Glu Ala Asp Asp AspGlu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 GAG GCT GAG GAACCC TAC GAA GAA GCC ACA GAG AGA ACC ACC AGC ATT 816 Glu Ala Glu Glu ProTyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 GCC ACC ACC ACCACC ACC ACC ACA GAG TCT GTG GAA GAG GTG GTT CGA 864 Ala Thr Thr Thr ThrThr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 GAG GTG TGC TCTGAA CAA GCC GAG ACG GGG CCG TGC CGA GCA ATG ATC 912 Glu Val Cys Ser GluGln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 TCC CGC TGG TACTTT GAT GTG ACT GAA GGG AAG TGT GCC CCA TTC TTT 960 Ser Arg Trp Tyr PheAsp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 TAC GGC GGATGT GGC GGC AAC CGG AAC AAC TTT GAC ACA GAA GAG TAC 1008 Tyr Gly Gly CysGly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 TGC ATG GCCGTG TGT GGC AGC GCC ATG TCC CAA AGT TTA CTC AAG ACT 1056 Cys Met Ala ValCys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350 ACC CAG GAACCT CTT GCC CGA GAT CCT GTT AAA CTT CCT ACA ACA GCA 1104 Thr Gln Glu ProLeu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 GCC AGT ACCCCT GAT GCC GTT GAC AAG TAT CTC GAG ACA CCT GGG GAT 1152 Ala Ser Thr ProAsp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380 GAG AAT GAACAT GCC CAT TTC CAG AAA GCC AAA GAG AGG CTT GAG GCC 1200 Glu Asn Glu HisAla His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400 AAG CACCGA GAG AGA ATG TCC CAG GTC ATG AGA GAA TGG GAA GAG GCA 1248 Lys His ArgGlu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410 415 GAA CGTCAA GCA AAG AAC TTG CCT AAA GCT GAT AAG AAG GCA GTT ATC 1296 Glu Arg GlnAla Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 CAG CATTTC CAG GAG AAA GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC 1344 Gln His PheGln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445 GAG AGACAG CAG CTG GTG GAG ACA CAC ATG GCC AGA GTG GAA GCC ATG 1392 Glu Arg GlnGln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 CTC AATGAC CGC CGC CGC CTG GCC CTG GAG AAC TAC ATC ACC GCT CTG 1440 Leu Asn AspArg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 CAGGCT GTT CCT CCT CGG CCT CGT CAC GTG TTC AAT ATG CTA AAG AAG 1488 Gln AlaVal Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495 TATGTC CGC GCA GAA CAG AAG GAC AGA CAG CAC ACC CTA AAG CAT TTC 1536 Tyr ValArg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510 GAGCAT GTG CGC ATG GTG GAT CCC AAG AAA GCC GCT CAG ATC CGG TCC 1584 Glu HisVal Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525 CAGGTT ATG ACA CAC CTC CGT GTG ATT TAT GAG CGC ATG AAT CAG TCT 1632 Gln ValMet Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540 CTCTCC CTG CTC TAC AAC GTG CCT GCA GTG GCC GAG GAG ATT CAG GAT 1680 Leu SerLeu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp 545 550 555 560GAA GTT GAT GAG CTG CTT CAG AAA GAG CAA AAC TAT TCA GAT GAC GTC 1728 GluVal Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575TTG GCC AAC ATG ATT AGT GAA CCA AGG ATC AGT TAC GGA AAC GAT GCT 1776 LeuAla Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590CTC ATG CCA TCT TTG ACC GAA ACG AAA ACC ACC GTG GAG CTC CTT CCC 1824 LeuMet Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605GTG AAT GGA GAG TTC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT 1872 ValAsn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620GGG GCT GAC TCT GTG CCA GCC AAC ACA GAA AAC GAA GTT GAG CCT GTT 1920 GlyAla Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635640 GAT GCC CGC CCT GCT GCC GAC CGA GGA CTG ACC ACT CGA CCA GGT TCT 1968Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650655 GGG TTG ACA AAT ATC AAG ACG GAG GAG ATC TCT GAA GTG AAG ATG GAT 2016Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665670 GCA GAA TTC CGA CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA TTG 2064Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680685 GTG TTC TTT GCA GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT GGA 2112Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695700 CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC TTG 2160Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710715 720 GTG ATG CTG AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG2208 Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725730 735 GAG GTT GAC GCC GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG2256 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740745 750 CAG CAG AAC GGC TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG2304 Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755760 765 CAG AAC 2310 Gln Asn 770 770 amino acids amino acid linearprotein 6 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr AlaArg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu AlaGlu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met AsnVal Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr CysIle Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr ProGlu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val ThrIle Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His ProHis Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val SerAsp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln GluArg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val AlaLys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp TyrGly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val GluPhe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp SerAla Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly AlaAsp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu ValAla Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu AlaAsp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 GluAla Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280285 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr GluGlu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser LeuLeu Lys Thr 340 345 350 Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys LeuPro Thr Thr Ala 355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr LeuGlu Thr Pro Gly Asp 370 375 380 Glu Asn Glu His Ala His Phe Gln Lys AlaLys Glu Arg Leu Glu Ala 385 390 395 400 Lys His Arg Glu Arg Met Ser GlnVal Met Arg Glu Trp Glu Glu Ala 405 410 415 Glu Arg Gln Ala Lys Asn LeuPro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 Gln His Phe Gln Glu LysVal Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445 Glu Arg Gln Gln LeuVal Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp ArgArg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 Gln AlaVal Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495 TyrVal Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520525 Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp545 550 555 560 Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser AspAsp Val 565 570 575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr GlyAsn Asp Ala 580 585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr ValGlu Leu Leu Pro 595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu GlnPro Trp His Ser Phe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr GluAsn Glu Val Glu Pro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala Asp ArgGly Leu Thr Thr Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys ThrGlu Glu Ile Ser Glu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His AspSer Gly Tyr Glu Val His His Gln Lys Leu 675 680 685 Val Phe Phe Ala GluAsp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val GlyGly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val MetLeu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735 GluVal Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755 760765 Gln Asn 770 20 base pairs nucleic acid single linear DNA NO NO 7CCGATGATGA CGAGGACGAT 20 20 base pairs nucleic acid single linear DNA NONO 8 TGAACACGTG ACGAGGCCGA 20 5 amino acids amino acid linear peptide 9Phe Arg Val Gly Ser 5 8 amino acids amino acid linear peptide 10 Asp AlaGlu Phe Arg Gly Gly Cys 5

We claim:
 1. A method for testing compounds for an effect on anAlzheimer's disease marker comprising a) administering the compound tobe tested to a non-human transgenic mammal, or mammalian cells derivedfrom the transgenic mammal, wherein the transgenic mammal has a nucleicacid construct stably incorporated into the genome, wherein theconstruct comprises a promoter for expression of the construct in amammalian cell and a region encoding an Aβ-containing protein, whereinthe promoter is operatively linked to the region, wherein the regioncomprises DNA encoding the Aβ-containing protein, wherein theAβ-containing protein consists of all or a contiguous portion of aprotein selected from the group consisting of APP770, APP770 bearing amutation in one or more of the amino acids selected from the groupconsisting of amino acid 669, 670, 671, 690, 692, and 717, APP751,APP751 bearing a mutation in one or more of the amino acids selectedfrom the group consisting of amino acid 669, 670, 671, 690, 692, and717, APP695, and APP695 bearing a mutation in one or more of the aminoacids selected from the group consisting of amino acid 669, 670, 671,690, 692, and 717, wherein the Aβ-containing protein includes aminoacids 672 to 714 of human APP, wherein the promoter mediates expressionof the construct such that Aβ_(tot) is expressed at a level of at least30 nanograms per gram of brain tissue of the mammal when it is two tofour months old, Aβ₁₋₄₂ is expressed at a level of at least 8.5nanograms per gram of brain tissue of the mammal when it is two to fourmonths old, APP and APPα combined are expressed at a level of at least150 picomoles per gram of brain tissue of the mammal when it is two tofour months old, APPβ is expressed at a level of at least 40 picomolesper gram of brain tissue of the mammal when it is two to four monthsold, and/or mRNA encoding the Aβ-containing protein is expressed to alevel at least twice that of mRNA encoding the endogenous APP of thetransgenic mammal in brain tissue of the mammal when it is two to fourmonths old; and detecting or measuring the Alzheimer's disease markersuch that any difference between the marker in the transgenic mammal, orby mammalian cells derived from the transgenic mammal, and the marker ina transgenic mammal, or by mammalian cells derived therefrom, to whichthe compound has not been administered, is observed, wherein an observeddifference in the marker indicates that the compound has an effect onthe marker.
 2. The method of claim 1 wherein the Aβ-containing proteinis selected from the group consisting of APP770; APP770 bearing amutation in the codon encoding one or more amino acids selected from thegroup consisting of amino acid 669, 670, 671, 690, 692, 717; APP751;APP751 bearing a mutation in the codon encoding one or more amino acidsselected from the group consisting of amino acid 669, 670, 671, 690,692, 717; APP695; APP695 bearing a mutation in the codon encoding one ormore amino acids selected from the group consisting of amino acid 669,670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770of APP; a protein consisting of amino acids 670 to 770 of APP; a proteinconsisting of amino acids 672 to 770 of APP; and a protein consisting ofamino acids 672 to 714 of APP.
 3. The method of claim 2 wherein the DNAencoding the Aβ-containing protein is cDNA or a cDNA/genomic DNA hybrid,wherein the cDNA/genomic DNA hybrid includes at least one APP intronsequence wherein the intron sequence is sufficient for splicing.
 4. Themethod of claim 1 wherein the promoter is the human platelet derivedgrowth factor β chain gene promoter.
 5. The method of claim 1 whereinthe region further comprises DNA encoding a second protein, wherein theDNA encoding the Aβ-containing protein and the DNA encoding the secondprotein are operative linked such that the region encodes anAβ-containing fusion protein comprising a fusion of the Aβ-containingprotein and the second protein.
 6. The method of claim 5 wherein thesecond protein is a signal peptide.
 7. The method of claim 1 wherein theAlzheimer's disease marker is a protein and the observed difference isan increase or decrease in the amount of the protein present in thetransgenic mammal, or in mammalian cells derived therefrom, to which thecompound has been administered.
 8. The method of claim 7 wherein theprotein is selected from the group consisting of Cat D,B, NeuronalThread Protein, nicotine receptors, 5-HT₂ receptor, NMDA receptor,α2-adrenergic receptor, synaptophysin, p65, glutamine synthetase,glucose transporter, PPI kinase, GAP43, cytochrome oxidase, hemeoxygenase, calbindin, adenosine A1 receptors, choline acetyltransferase,acetylcholinesterase, glial fibrillary acidic protein (GFAP),α1-antitrypsin, C-reactive protein, α2-macroglobulin, IL-1α, IL-1β,TNFα, IL-6, HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4,CD45, CD64, CD4, spectrin, tau, ubiquitin, MAP-2, apolipoprotein E,nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),advanced glycosylation end products, receptor for advanced glycosylationend products, COX-2, CD18, C3, fibroblast growth factor, CD44, ICAM-1,lactotransferrin, C1q, C3d, C4d, C5b-9, gamma RI, Fc gamma RII, CD8,CD59, vitronectin, vitronectin receptor, beta-3 integrin, Apo J,clusterin, type 2 plasminogen activator inhibitor, midkine, macrophagecolony stimulating factor receptor, MRP14, 27E10, interferon-alpha,S100β, cPLA₂, c-jun, c-fos, HSP27, HSP70, MAP5, membrane lipidperoxidase, protein carbonyl formation, junB, jund, fosB, fra1, cyclinD1, p53, NGFI-A, NGFI-B, IκB, NFκB, IL-8, MCP-1, MIP-1α, matrixmetaloproteinases, 4-hydroxynonenal-protein conjugates, amyloid Pcomponent, laminin, and collagen type IV.
 9. The method of claim 1wherein the Alzheimer's disease marker is a protein and the observeddifference is a reduction or absence of the protein in plaques orneuritic tissue present in the transgenic mammal to which the compoundhas been administered.
 10. The method of claim 9 wherein the protein isselected from the group consisting of Cat D,B, protein kinase C, NADPH,C3d, C1q, C5, C4bp, C5a-C9, tau, ubiquitin, MAP-2, neurofilaments,heparin sulfate, chrondroitin sulphate, apolipoprotein E, nerve growthfactor (NGF), brain-derived neurotrophic factor (BDNF), glycosylationend products, amyloid P component, laminin, and collagen type IV. 11.The method of claim 1 wherein the Alzheimer's disease marker is aprotein and the observed difference is an increase or decrease in theenzymatic or biochemical activity of the protein in the transgenicmammal, or in mammalian cells derived therefrom, to which the compoundhas been administered.
 12. The method of claim 11 wherein the protein isselected from the group consisting of nicotine receptors, 5-HT₂receptor, NMDA receptor, α2-adrenergic receptor, glutamine synthetase,glucose transporter, PPI kinase, cytochrome oxidase, heme oxygenase,calbindin, adenosine A1 receptors, choline acetyltransferase,acetylcholinesterase, glial fibrillary acidic protein (GFAP),α1-antitrypsin, C-reactive protein, α2-macroglobulin, IL-1, TNFα, IL-6,HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64,CD4, spectrin, ubiquitin, and apolipoprotein E.
 13. The method of claim1 wherein the Alzheimer's disease marker is a nucleic acid encoding aprotein and the observed difference is an increase or decrease in theamount of the nucleic acid present in the transgenic mammal, or inmammalian cells derived therefrom, to which the compound has beenadministered.
 14. The method of claim 13 wherein the encoded protein isselected from the group consisting of growth inhibitory factor, Cat D,B,Neuronal Thread Protein, nicotine receptors, 5-HT₂ receptor, NMDAreceptor, α2-adrenergic receptor, synaptophysin, p65, glutaminesynthetase, glucose transporter, PPI kinase, GAP43, cytochrome oxidase,heme oxygenase, calbindin, adenosine A1 receptors, cholineacetyltransferase, acetylcholinesterase, glial fibrillary acidic protein(GFAP), α1-antitrypsin, C-reactive protein, α2-macroglobulin, IL-1,TNFα, IL-6, HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4,CD45, CD64, CD4, spectrin, tau, ubiquitin, MAP-2, apolipoprotein E,nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),advanced glycosylation end products, receptor for advanced glycosylationend products, COX-2, CD18, C3, fibroblast growth factor, CD44, ICAM-1,lactotransferrin, C1q, C3d, C4d, C5b-9, gamma RI, Fc gamma RII, CD8,CD59, vitronectin, vitronectin receptor, beta-3 integrin, Apo J,clusterin, type 2 plasminogen activator inhibitor, midkine, macrophagecolony stimulating factor receptor, MRP14, 27E10, interferon-alpha,S100β, cPLA₂, c-jun, c-fos, HSP27, HSP70, MAP5, membrane lipidperoxidase, protein carbonyl formation, junB, junD, fosB, fra1, cyclinD1, p53, NGFI-A, NGFI-B, IκB, NFκB, IL-8, MCP-1, MIP-1α, matrixmetaloproteinases, 4-hydroxynonenal-protein conjugates, amyloid Pcomponent, laminin, and collagen type IV.
 15. The method of claim 1wherein the Alzheimer's disease marker is a behavior and the observeddifference is a change in the behavior observed in the transgenic mammalto which the compound has been administered.
 16. The method of claim 15wherein the behavior is selected from the group consisting of behaviorusing working memory, behavior using reference memory, locomotoractivity, emotional reactivity to a novel environment or to novelobjects, and object recognition.
 17. The method of claim 1 wherein theAlzheimer's disease marker is a histopathology and the observeddifference is a decrease in the extent or severity of the histopathologypresent in the transgenic mammal to which the compound has beenadministered.
 18. The method of claim 17 wherein the histopathologymarker is selected from the group consisting of compacted plaques,neuritic dystrophy, gliosis, Aβ deposits, decreased synaptic density,and neuropil abnormalities.
 19. The method of claim 1 wherein theAlzheimer's disease marker is cognition and the observed difference is achange in the cognition of the transgenic mammal to which the compoundhas been administered.
 20. The method of claim 1 wherein the marker isdetected or measured using RT-PCR, RNase protection, Northern analysis,R-dot analysis, ELISA, antibody staining, laser scanning confocalimaging, and immunoelectron micrography.
 21. The method of claim 1wherein the mammals are rodents.
 22. The method of claim 1 wherein thecodon encoding amino acid 717 is mutated to encode an amino acidselected from the group consisting of Ile, Phe, Gly, Tyr, Leu, Ala, Pro,Trp, Met, Ser, Thr, Asn, and Gln.
 23. The method of claim 22 wherein thecodon encoding amino acid 717 is mutated to encode Phe.
 24. The methodof claim 1 wherein the codon encoding amino acid 670 is mutated toencode an amino acid selected from the group consisting of Asn and Glu,or the codon encoding amino acid 670 is deleted, and/or wherein thecodon encoding amino acid 671 is mutated to encode an amino acidselected from the group consisting of Ile, Leu, Tyr, Lys, Glu, Val, andAla, or the codon encoding amino acid 671 is deleted.
 25. The method ofclaim 24 wherein the codon encoding amino acid 670 is mutated to encodeAsn, and/or the codon encoding amino acid 671 is mutated to encode Leuor Tyr.
 26. The method of claim 1 wherein the promoter mediatesexpression of the construct such that Aβ_(tot) is expressed at a levelof at least 30 nanograms per gram of hippocampal or cortical braintissue of the mammal when it is two to four months old, Aβ₁₋₄₂ isexpressed at a level of at least 8.5 nanograms per gram of hippocampalor cortical brain tissue of the mammal when it is two to four monthsold, APP and APPα combined are expressed at a level of at least 150picomoles per gram of hippocampal or cortical brain tissue of the mammalwhen it is two to four months old, APPβ is expressed at a level of atleast 40 picomoles per gram of hippocampal or cortical brain tissue ofthe mammal when it is two to four months old, and/or mRNA encoding theAβ-containing protein is expressed to a level at least twice that ofmRNA encoding the endogenous APP of the transgenic mammal in hippocampalor cortical brain tissue of the mammal when it is two to four monthsold.
 27. The method of claim 1 wherein amyloid plaques that can bestained with Congo Red are present in brain tissue of the mammal.